Alu retrotransposon quantification in the retina and plasma: Mechanism-based risk assessment in age-related macular degeneration

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Abstract

Purpose

Alu RNAs are endogenous pathogens in age-related macular degeneration (AMD). Currently, efforts to develop diagnostic and prognostic tools are limited. Yet, because AMD is a chronic disease, tools to detect pre-clinical disease and monitor progression are an essential part of an effective and comprehensive therapeutic strategy. We tested plasma Alu RNA as a potential biomarker for AMD. Also, cells can convert Alu RNA into DNA, and as such, we determined Alu DNA content of RPE cells. We also genotyped RPE cells for a panel of known AMD risk single nucleotide polymorphisms, and developed an AMD risk stratification system based on age, genotype, and Alu DNA content.

Methods

RPE cells were isolated from human donor eyes were harvested within 5-10 hours after death. RPE cells were grown in cell culture for four passages. DNA was extracted and subjected to genotyping for AMD-associated SNPs using TaqMan probes. Plasma sample RNA was extracted with Trizol LS and reverse transcribed. qPCR for DNA performed with standard curves of Alu-specific primers and housekeeping single copy genes; for cDNA, Alu quantified per mL plasma.

Results

The RPE of AMD donors contain increased quantities of Alu DNA. Our results suggest that Alu DNA content increases as a function of age and disease status, and that genotype also modulates disease risk. Finally, Alu RNA content in plasma was significantly increased in AMD versus age-matched healthy controls.

Conclusion

These efforts set the stage for the utilization of plasma Alu RNA quantity as a novel mechanism-based biomarker in AMD, and also implicate increased Alu DNA burden as a potential mediator of Alu RNA-induced RPE cell death in AMD.

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