Whole exome sequencing of patients with retinal degenerative disease To identify genetic mutations that cause retinal degeneration, we have analyzed over 200 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. Genetics of AMD Age-related macular degeneration (AMD) is a leading cause of blindness in the developed world. While many AMD susceptibility variants have been identified, their influence on AMD progression has not been elucidated. We were involved in several different projects, as follows. Using data from two large clinical trials, Age-Related Eye Disease Study (AREDS) and AREDS2, we evaluated the effects of 34 known risk variants on disease progression. We show that for prediction of AMD progression, addition of GRS to the demographic/environmental risk factors considerably improved the prediction performance. Our model for predicting the disease progression risk demonstrated satisfactory performance in both cohorts, and we recommend its use with baseline AMD severity scores plus baseline age, education level, and smoking status, either with or without GRS (PMID 28341650). We are now extending this analysis to genome-wide bivariate time-to-event test for AMD progression with 9 million variants on 2,721 AREDS participants. 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. To discover additional rare variants and characterize the GWAS locus further, we performed whole genome sequencing of 2,394 cases and 2,393 controls and the analysis is in progress. We have looked into the contribution of rare variants in GWAS loci of sub-types of AMD. We have also participated in a deep phenotype association study in AREDS2 reveals specific phenotype association with genetic variants involved in AMD. This shows the association of the SNP at the ARMS2/HTRA1 locus with subretinal/sub-RPE hemorrhage and poorer visual acuity and of SNPs at CFH locus with drusen area may provide new insights in pathophysiological pathways underlying different stages of AMD. We also generated WES data in 19 large multigenerational AMD families to ascertain high-penetrance causative allele(s). To enhance the power of analysis, we are collaborating with other groups to extract useful information. Functional Genomic Analysis of AMD: Current variants/loci can explain 50-60% of AMD heritability, with ARMS2 and CFH accounting for bulk of the effect. Our collaborative studies suggest increased retinal mitochondrial DNA damage in patients with CFH risk alleles (Ferrington et al. 2016). We are now focusing on dissection of relative contributions of variants, genes and pathways to AMD pathology. A Reference Transcriptome of Adult Human Retina: We have generated a comprehensive reference transcriptome of the normal human retina by RNA-seq analysis of 105 healthy donor samples (from Dr. D Ferrington) We have characterized both annotated coding regions as well as un-annotated transcripts using de novo assembly. This dataset would be valuable for the vision community while designing interventions for retinal diseases. Gene and Pathways Associated with AMD Progression: We performed transcriptomic studies of 390 donor AMD retinas (197 early, 127 intermediate, 66 advanced). 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. A comprehensive resource of eQTL in retina: interpretation of the GWAS findings, and application in risk evaluation and therapeutic interventions remains one of the major challenges for researchers. A large proportion GWAS signals reside in the non-coding region and thus underlying genes/variants are not conspicuous and are most likely to mediate their effect through regulation. To understand this regulation, we used the DNA from 523 donor subjects and genotyped 603,583 markers using UM HUNT Biobank v1.0 chip then obtained the inferred genotypes for 6,554,241 variants using 1000G reference panel through imputation. After adjusting for LD structure, this resulted in 41,364 cis and 8,165 trans eQTL, several of which are in AMD GWAS loci and can help elucidate the causal variants. Identifying common molecular pathways in various retinal degeneration mice models through characterization of gene expression trends in rod photoreceptors A major cause for progressive retinal degenerative diseases is rod photoreceptor cell death, eventually leading to loss of the photoreceptor layer, resulting in loss of vision. In mice, several mutants have been identified or developed that closely mimic human RDD. The pathological presentation of these mutants also differ greatly, with some showing very early onset and rapid degeneration of photoreceptor layer in less than 30 days post birth (rd1), while in others photoreceptors survive for several months (rds). Since in several of these model systems, disease onset overlaps with development, it is difficult to ascertain the actual molecular changes associated with pathology. Extensive transcriptomic profiling using next generation sequencing and analysis, suggest massive irregularities in development trajectories in photoreceptors of disease models, demonstrating that rod cells experience changes much before cells are set to apoptosis. In particular, photoreceptors from RD mice retina show abnormal transcriptomic dynamics between postnatal day (P)6 and P8, which as described previously by our group is a hallmark of wt rod maturation. Further investigations highlight major changes in mitochondrial metabolism and the ATP generation pathway via oxidative phosphorylation (oxphos). Mitochondria play an essential role in stringently regulating energy homeostasis, cell signaling and survival. Retinal photoreceptors have high oxygen demand and are especially vulnerable to mitochondrial dysfunction, which has been implicated as a causal factor of neuronal cell death in retinal diseases. 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 bioinformtic analysis of transcriptomic and proteomic data from rd1 and wt retina support the notion of dysfunctional oxphos and mitochondrial metabolism, as also seen from direct OCR measurement. 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. Furthermore, we used bioinformatics tools to evaluate transcriptomic and proteomic profiles of mutant photoreceptors/retinas in rd1 mice to determine genes or pathways underlying mitochondrial dysfunction and photoreceptor death.
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