Patterns of gene expression and allele-specific expression vary among macular tissues and clinical stages of Age-related Macular Degeneration

Age-related macular degeneration (AMD) is a complex neurodegenerative disease and is the leading cause of blindness in the aging community. AMD progresses, has an early and intermediate stages (both dry and two late stages, neovascular and geographic atrophy (GA). It is the late stages that are responsible for the majority of visual impairment and blindness. Available medicines are directed against the less common wet form and do not cure or reverse vision loss. Therefore, it is imperative to identify preventive and therapeutic targets. As the mechanism for AMD is unclear, one, way is to interrogate well characterized disease affected tissue in the appropriate geographic region that is the macular neural retina and macular retina pigment epithelium (RPE)/choroid. We investigated differential gene expression (DEG) across the clinical stages of AMD in the macula of the primary affected tissue compared to well characterized normal using a standardized published protocol (Owen et al., 2019). Donor eyes (n=27) were Caucasian with an age range of 60-94 and 63% were male, and tissue from the macula RPE/choroid and macula neural retina were taken from the same eye. Donor eyes were recovered within 6 hours post mortem interval time and phenotyped by ophthalmic experts using multi modal imaging (fundus photos and SD-OCT) to ensure maximal preservation of RNA quality and accuracy of diagnosis. Utilizing DESeq2, followed PCA, Benjamini Hockberg Analysis to control for the FDR, Bonferonni for the paired comparisons: a total of 26,650 genes were expressed in the macula RPE/choroid and/or macula retina with 1,204 genes found to be statistically different between neovascular AMD and normal eyes, 40 genes found between intermediate AMD (AREDS3) and normal eyes, and 1,194 genes found between intermediate AMD and neovascular AMD. A comparison of the DEGs from intermediate AMD with normal eyes and neovascular AMD with normal eyes showed an overlap of six genes: MTRNR2L1, CLEC2L, CCM2L, CYP4X1, GLDN, and SMAD7. The top pathways of DEGs identified through Ingenuity Pathway Analysis (IPA) for intermediate AMD with normal eyes were the interferon signaling and Th1 and Th2 activation pathways, while for neovascular AMD with normal eyes it was the phototransduction and SNARE signaling pathways. Allele specific expression (ASE) revealed known coding regions in previously reported GWAS loci demonstrated that significant ASE for C3 rs2230199 and CFH rs1061170 occurred in the macula RPE/choroid for normal, and intermediate AMD while ASE for CFH rs1061147 occurred in occurred in the macula RPE/choroid for normal, and intermediate and neovascular AMD. An investigation of previously identified GWAS loci (Fritsche et al., 2016) revealed that 76% of the 34 loci previously identified were significantly differentially expressed between normal macular RPE/choroid and macular neural retina, with 77% of these identified loci expressed higher in the RPE. Pathway analysis was conducted on these DEGs to reveal the involvement of pathways previously implicated in AMD as well as being the first study to assess ASE across the clinical spectrum of AMD. By utilizing a systems biology approach that integrates clinical, experimental, genomic, and phenotypic data to aid in the understanding of the pathogenesis of AMD.