Functional Genomic Analysis of AMD: The variants/loci that are currently known can explain 50-60% of AMD heritability, with ARMS2 and CFH accounting for bulk of the effect. Our collaborative genome-wide association studies (GWAS) have identified 52 variants at 34 genetic loci associated with AMD. We have designed studies to understand how associated genetic variations especially in the non-coding regions of the genome contribute to AMD. We performed extensive transcriptome, and eQTL analysis of human peripheral retina and identified target susceptibility genes at 6 previously AMD-associated GWAS loci and three novel AMD target genes. In order to functionally characterize the variant-gene relationship, we are performing in-vitro luciferase assays of genetic variants and producing CRISPER-Cas9 based single/double knock-out mice of target genes. We have generated knockout mice for 6 genes and initiated their characterization. Our combined approach of in-vitro/in-vivo studies will help in understanding the functions of variants identified in non-coding regions of AMD-associated GWAS loci. We have also performed transcriptomic studies of 150 postmortem donor AMD maculas. Differential expression, pathway and co-expression analysis has revealed the core transcriptome signature and pathways dysregulated in AMD, providing several novel candidates and insights into the AMD pathobiology. To discover additional rare variants and characterize the GWAS locus further, we performed whole genome sequencing of 2,394 cases and 2,393 controls in collaboration with Drs. Chew and scientists at the University of Michigan, who are analyzing the data. In collaboration with Dr. Chew, we have also participated in a deep phenotype association study in AREDS2. This study showed the association of SNPs at the ARMS2/HTRA1 locus with subretinal/sub-RPE hemorrhage and poorer visual acuity and of SNPs at CFH locus with drusen. We also participated in studying the genetic pleiotrophy between AMD with other complex diseases. We demonstrate a substantial overlap of the genetics of several complex diseases/traits with AMD and provide statistically significant evidence for an additional 20 loci associated with AMD. This highlights the possibility that so far unrelated pathologies may have disease pathways in common. We have generated whole exome sequencing data from 19 large multigenerational AMD families from Univ of Michigan to ascertain high-penetrance causative allele(s). To enhance the power of analysis, we are collaborating with other groups to extract useful information. Epigenetic changes in AMD: The epigenome, as characterized primarily by DNA and histone modifications, plays an important role in regulating gene/protein expression, especially during aging and disease. We performed genome wide DNA methylation profiles of 40 control, 26 early AMD, and 26 advanced AMD retinas using the Infinium MethylationEPIC BeadChip. Predicted age from Horvaths epigenetic clock model were decades younger than biological age, suggesting that the retina has enhanced epigenetic maintenance machinery compared to other tissues. No significant global differences were detected in gene expression between control and AMD retinas. Differential methylation analysis using surrogate variables to control for covariates showed no differentially methylated loci in AMD compared to control. Colocalization of eQTLs, mQTLs, and reported AMD loci indicate that the glutathione metabolism pathway may be implicated in aging, and probably in AMD. Whole exome sequencing of patients with retinal degenerative disease: To identify genetic mutations that cause retinal degeneration, we have analyzed over 400 individuals (patients and unaffected controls) from various clinical collaborators in the US and worldwide. Using whole genome sequencing, we have identified a novel inherited retinal disease gene, IDH3A in 4 unrelated families as a cause of Retinitis Pigmentosa. Further data analysis is in progress. Additionally, we have also identified several known disease-causing variants in ABCA4 and other genes known to cause other retinal degenerative diseases. We can estimate that about 50% of these cases have been solved, in that we now know the variants responsible for the disease. We are still in the process completing the analysis. Identifying common molecular pathways in retinal degeneration: Transcriptomic profiling using next generation sequencing suggested irregularities in development trajectories of photoreceptors in the disease models, demonstrating that rod cells experience changes much before their phenotypic changes. Our studies identified major changes in mitochondrial metabolism and the ATP generation via oxidative phosphorylation (oxphos). We assayed the physiological function of mitochondria by directly measuring oxygen consumption rate (OCR) and lactate production in ex vivo retina of wild type and retinal degeneration mouse models. Integrative bioinformatic analysis of transcriptomic, proteomic and metabolomic data from rd1 and wt retina support the notion of dysfunctional oxphos and mitochondrial metabolism. We plan to continue investigating the upstream regulators of oxphos while extending the integrative analysis to all models, with the objective to identify common drug targets to arrest photoreceptor death in RDD. Heat shock proteins are believed to play a pro-survival role in degenerating neurons. We show that expression of stress activated heat shock family member HSP70 is elevated, at early stage, in the retina of different retinal degeneration mouse models. We tested the hypothesized pro-survival role of heat shock protein by over expressing HSP70, driving by Crx promoter, in the retina of different retinal degeneration mouse models. We observed that the effect of elevated HSP70 expression varies depending on the nature of altered biological processes in different models. Our data suggests that increased HSP70 expression might be beneficial as potential therapeutic method in some forms of retinal disease.
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