Abstract
Purpose :
Age-related dysfunction and/or death of retinal pigment epithelial (RPE) cells have been associated with various eye diseases, such as age-related macular disorders (AMD). Identifying age-related gene expression changes in the RPE may help shed light on pathogenic mechanisms that affect age-related vision loss or blindness.
Methods :
RNA was collected from mouse RPE at 4 different time points of ages: 6 months (M), 12 M, 18 M, and 21 M. Mice, all male and the same genotype background, were kept on identical diet and light exposure regimes. PolyA RNA-Sequencing was performed on three-four mouse RPE replicates per age group. An un-stranded, paired end (PE) library preparation was performed. Samples were multiplexed on the Illumina’s sequencer for 75 cycles. An average sequencing coverage of 22.3 million high-quality reads was achieved across all replicates and age groups. The STAR aligner was used to align PE reads and featureCount was used to quantify gene expression. SmartSVA was applied to account for hidden confounding factors on gene expression and normalize between samples. To perform differential gene expression, we used a combination of two programs: ImpulseDE2 and DESeq2. ImpulseDE2 implements an impulse-based model to capture “monotone” and “transient” changes across the time points. The likelihood-ratio test (LRT) implemented in DESeq2 was applied to compare among all four time point combinations. Genes with Benjamini-Hochberg adjusted P-value <0.05 in both methods were considered a set of high confidence genes whose expression changes with age. g:Profiler was used for enrichment of transcription factor (TF) regulation.
Results :
There were 352 high confidence genes (q<0.05), whose expression significantly increased (average 5-fold) or decreased (average 8.3-fold) between 6 M to 21 M in mouse RPE. These genes formed five clusters with different patterns of up or downregulation in early and late ages. The 352 genes were enriched for targets of 111 TFs (q<0.05), including ZF5 found to be associated with Stargardt disease and E2F transcription factor 1 (E2F1) associated with AMD.
Conclusions :
We present a new methodological process for identifying genes differentially expressed across different time points with RNA-Seq data. In addition, this study identified age-related genes in mouse RPE that can be used to further investigate the role of aging in eye disease.
This is a 2021 ARVO Annual Meeting abstract.