March 2012
Volume 53, Issue 14
Free
ARVO Annual Meeting Abstract  |   March 2012
Conjunctival Gene Expression Patterns in Dry Eye; Real Time PCR and Beyond
Author Affiliations & Notes
  • John L. Bradley
    Vision Sciences,
    Univ of Alabama at Birmingham, Birmingham, Alabama
  • Keshia S. Elder
    College of Optometry, UMSL, St Louis, Missouri
  • Tammy P. Than
    School of Optometry,
    Univ of Alabama at Birmingham, Birmingham, Alabama
  • Roderick J. Fullard
    Vision Sciences,
    Univ of Alabama at Birmingham, Birmingham, Alabama
  • Footnotes
    Commercial Relationships  John L. Bradley, None; Keshia S. Elder, None; Tammy P. Than, None; Roderick J. Fullard, None
  • Footnotes
    Support  Eyesight Foundation of Alabama Grant FY2010-11-226
Investigative Ophthalmology & Visual Science March 2012, Vol.53, 538. doi:
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      John L. Bradley, Keshia S. Elder, Tammy P. Than, Roderick J. Fullard; Conjunctival Gene Expression Patterns in Dry Eye; Real Time PCR and Beyond. Invest. Ophthalmol. Vis. Sci. 2012;53(14):538.

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

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Abstract

Purpose: : While the DEWS report confirms that there are multiple causes of dry eye there is a clear lack of FDA-approved dry-eye treatments. To help us develop new treatments we need to better understand how inflammatory patterns differ among dry eye types. Predictive modeling of biomarkers has the potential to better characterize dry eye types. This study investigates the viability of conjunctival impression cytology and gene expression analysis to achieve this goal.

Methods: : Twenty-five subjects including dry eye and normals participated in the study. After a clinical dry-eye evaluation, impression cytology (Millipore HAWP filter discs) was used to collect up to eight conjunctival cell samples per eye. The discs were immediately transferred to lysing solution and RNA was subsequently isolated using a Qiagen RNeasy Plus Mini Kit. An Applied Biosystems (AB) High Capacity RNA-to-cDNA Kit was used for reverse transcription. Four samples were then applied to a 384-well TaqMan low density array (TLDA) card containing 96 custom selected genes per sample. Samples were analyzed using an AB 7900HT Fast Real-Time PCR System.

Results: : While the focus of this study is TLDA PCR results, extracted RNA was of sufficient quantity and quality to enable genome-wide assay (e.g. Illumina HT12 chip) or transcriptome analysis. The TLDA card method was sensitive enough to detect gene expression differences among conjunctival anatomical sites. When comparing the nasal to temporal conjunctiva, MCP-1 and MUC5AC were expressed at significantly higher levels in the nasal conjunctiva. Additionally, MUC5AC, CCR3, FGF2, IL-18R1, IL-15, and MIP-3β were expressed at significantly higher levels in the nasal conjunctiva when compared to the superior conjunctiva. Dividing patients according to Schirmer test results, the sub 10mm/5min group elicited lower expression of IP-10, MIG, IL-10, IL-7R, MUC5AC, IL-8, and IL-12p40. Significant correlations were also found based on other clinical results. There was a trend (although non-statistically significant) towards lower MUC-1 expression with increasing Lissamine and fluorescein ocular surface staining scores.

Conclusions: : While this study involves normals and primarily moderately dry eye patients, the fact that several gene patterns emerged demonstrates the potential of this technique, in particular when applied to more severe dry eye. Larger and more homogenous dry eye patient groups should significantly improve predictive modeling of dry eye. This may ultimately lead to better cause-directed treatments.

Keywords: cornea: tears/tear film/dry eye • gene/expression • conjunctiva 
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