June 2015
Volume 56, Issue 7
Free
ARVO Annual Meeting Abstract  |   June 2015
Transcriptome-wide analysis of gene expression in normal and age-related macular degeneration (AMD)
Author Affiliations & Notes
  • Venkata R M Chavali
    Ophthalmology, University of Pennsylvania, Philadelphia, PA
  • Harini Venkata Gudiseva
    Ophthalmology, University of Pennsylvania, Philadelphia, PA
  • Anita Bowman
    Ophthalmology, University of Pennsylvania, Philadelphia, PA
  • David W Collins
    Ophthalmology, University of Pennsylvania, Philadelphia, PA
  • Ahmad M Khalil
    Department of Genetics, Case Western Reserve University, Cleveland, OH
  • Joan M O'Brien
    Ophthalmology, University of Pennsylvania, Philadelphia, PA
  • Dwight Stambolian
    Ophthalmology, University of Pennsylvania, Philadelphia, PA
  • Footnotes
    Commercial Relationships Venkata Chavali, None; Harini Gudiseva, None; Anita Bowman, None; David Collins, None; Ahmad Khalil, None; Joan O'Brien, None; Dwight Stambolian, None
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science June 2015, Vol.56, 816. doi:
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      Venkata R M Chavali, Harini Venkata Gudiseva, Anita Bowman, David W Collins, Ahmad M Khalil, Joan M O'Brien, Dwight Stambolian; Transcriptome-wide analysis of gene expression in normal and age-related macular degeneration (AMD). Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):816.

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

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Abstract

Purpose: To identify differential expression of genes and long non-coding RNAs (lncRNA) in peripheral retina (PR) and peripheral retinal pigment epithelium-choroid (PRPC) tissues obtained from normal and age-related macular degeneration (AMD) donor eyes.

Methods: Cadaver eye globes from 8 normal and 8 AMD Caucasian donors, < 6-8hrs post mortem were freshly enucleated and preserved in RNAlater solution. For each globe, 8mm punches of the posterior eye were separated into PR and PRPC and their RNA isolated using AllPrep RNA/DNA mini kit (Qiagen). Sequencing libraries were prepared using the TruSeq Stranded Total RNA Sample Prep Kit and sequenced on the HiSeq 2000 (Illumina) generating 100bp paired end reads. Transcriptome analysis on combined sequencing data was done using bioinformatics analysis pipeline (including Tophat, Cuffdiff, MATS, Cufflinks and KOBAS), for read mapping, identification of differential alternative splicing (DAS) events, ab initio transcript assembly, lncRNA expression quantification, Gene Ontology (GO) and pathway enrichment analysis.

Results: DAS analysis yielded differences between layers and diseased states. We found 11478 DAS events between AMD PR vs PRPC, 22108 DAS events between normal PR vs PRC, 6805 DAS events between normal PRPC vs AMD PRCS and 6564 DAS events between normal PR vs AMD PR samples. We identified 11891 differentially expressed (DE) gene transcripts between normal PR vs PRCS, and 9654 DE transcripts between AMD PR vs PRCS tissues. Only 184 and 233 DE transcripts were found between normal PR vs AMD PR and normal PRCS vs AMD PRCS, respectively. The DE genes were significantly enriched for visual transduction pathways and retina homeostasis pathways in KEGG, GO analysis for comparison between normal PRCS and AMD PRCS. Extracellular matrix interactome and complement cascade pathways were enriched in both KEGG and GO pathways in comparison between normal and AMD PR samples. Cufflinks identified a total of 30,212 lncRNA candidates. We identified 17 and 11 DE lncRNA between normal PR vs AMD PR and normal PRCS vs AMD PRCS, respectively.

Conclusions: Several differentially expressed genes and lncRNA in PR and PRCS tissues in both normal and AMD diseased states were identified. Characterizing their function and role in protein interacting pathways will enable us to identify novel cellular pathways that are necessary for retinal homeostasis and those which lead to AMD.

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