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Dwight Stambolian, Christine A Curcio, David Cho, James A. Kimble, Jeffrey D Messinger, Randy Zauhar, Mingyao Li; Complement genes are differentially expressed in late age-related macular degeneration (AMD) and only in the neurosensory retina. Invest. Ophthalmol. Vis. Sci. 2016;57(12):No Pagination Specified. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Genome wide association studies (GWAS) for AMD has identified over 35 risk loci yet most of the causative genes have yet to be identified. Genomic convergence, an approach that combines different kinds of genomic data to prioritize genes could assist in their identification. We hypothesize that identifying the differences in the transcriptome of normal and diseased eyes along with GWAS will assist in identifying causative genes.
Postmortem human eyes (N=15) were recovered within 6 hours of death. One eye was preserved with RNAlater while the fellow eye was used for histology. cDNA libraries were made from the retina (R) and RPE/choroid/sclera (RPCS) of each eye (8 normal, 7 AMD) and sequenced on an Illumina HiSeq machine. We calculated the expression levels of 23,569 RefSeq protein-coding genes using the Fragments Per Kilobase of gene per Million mapped fragment (FPKM) metric. Genes considered differentially expressed (DE) had false discovery rate (FDR) p-value is <0.05.
Sequencing depth for each sample was >110 million paired-end reads. When comparing R in normal and AMD eyes, we found 374 DE genes. The most significant genes included APOD (p<0.00005,140-fold change) and complement cascade genes (i.e. CFH, p<0.00005, 3-fold change) among others. Comparing RPCS of normal and AMD eyes, we found 119 DE genes. The most DE genes in RPCS included CRYAA (p<0.00005, 25-fold change) and PDE6H (p<0.00005,17-fold change) among others. Comparison of normal eyes and AMD phenotypes (GA, neovascular and early) showed distinct differences in DE between layers and phenotypes. Specifically, very little DE was found for the complement cascade in RPCS for all AMD phenotypes. In the R layer, in contrast, CFH and CFD (p<0.00005,13-fold change) were DE in GA R while C1Q (p<0.00005,5-fold change) and CFI (p=0.00025, 2.5-fold change) were DE in neovascularization. Overlap between DE genes and GWAS loci identified 29 genes.
Our results support our hypothesis that utilizing DE analysis between normal and AMD eyes will assist in identifying causative genes. Twenty-two DE genes from R and 7 genes from RPCS map to GWAS loci. The specificity of retina layer for complement over-expression in AMD is striking. Additionally, complement DE was not present in early AMD but was in end-stage AMD suggesting that complement is not a cause of early AMD.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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