Allele-Specific Expression in Normal and Diseased Donor Eyes Reveals Potential Therapeutic Targets for Intermediate AMD

Abstract

Purpose : Age-related macular degeneration (AMD) is considered one of the most well genetically defined complex disorders; however, the mechanisms underlying disease pathophysiology are poorly understood. Current therapies address advanced AMD when vision loss is largely irreversible. Interventions directed towards earlier disease stages are desperately needed to prevent AMD blindness. Our analysis of allele-specific expression (ASE) profiles in AMD diseased tissues offers a robust approach toward identification of candidate gene and gene variants relevant to local pathophysiology and environmental risk.

Methods : Well characterized donor eye tissue (postmortem time of less than 6 hours) of 14 patients over the age of 70 years (6 with intermediate stage AMD and 8 healthy age-matched controls) were surveyed using whole exome sequencing, RNA sequencing and whole genome genotyping using illumina chips. To generate ASE profiles, Exome Seq Fastq data was processed and merged with SNP chips and RNA sequencing data to build “personal” alignment indexes using Lofreq and STAR. Using these alignments, differential gene analysis was performed on high quality SNPs to calculate an ensemble prioritization score for each gene using the data from the imprinted gene, eQTL and GTEx databases. ASE candidates were then selected given a specific prioritization score cutoff which was compared to the prioritization score distribution of known AMD genes and genes not associated with AMD using a Kolmogorov-Smirnov test. Validation of identified candidate genes was perfomed using drop-seq PCR of macular neural retina and RPE/choroid from AMD patients versus normal patient tissues.

Results : After initial identification using our novel pipleline and subsequent validation as described, our analysis identified three novel genes, EOC1, FMN1 and TF, to be statistically associated with intermediate AMD within the local diseased tissues. Statistical analysis using a Kolmogorov-Smirnov test further showed a significant difference between the distributions, p < 0.05.

Conclusions : Using our ASE pipeline to identify disease mechanisms could pinpoint novel therapeutic targets for intermediate AMD with relevance to local disease mechanisms and therefore, potential to slow or stop disease progression.

This is a 2021 ARVO Annual Meeting abstract.

 

Kolmogorov–Smirnov test on the distributions of prioritization scores of AMD genes and non-AMD genes