Shannath Merbs - US grants

melanoma; eye neoplasms; neoplasm /cancer genetics; uvea disorder; molecular oncology; loss of heterozygosity; genetic markers; choroid uvea; methylation; genetic mapping; tumor suppressor genes; gene deletion mutation; nucleic acid sequence; human tissue; polymerase chain reaction;

DESCRIPTION (provided by applicant): Retinal development, normal function, and disease are controlled, to a large extent, by the pattern of genes expressed by the cells of the retina. Gene expression can be modified by the local chromatin configuration of a gene. DNA methylation and histone modification, known as epigenetic modifications, can effect gene expression, and although they can be persistent and heritable, they do not directly change the primary DNA sequence. Despite the significant advances being made in understanding the role of epigenetics in gene regulation in other fields, little is known about the relationship between DNA methylation and tissue-specific gene expression in the developing and adult retina. Our preliminary studies show that, in the adult mouse, the region around the transcription start site of murine genes Rbp3 (PR-specific) and Rho (rod-specific) is hypomethylated in expressing photoreceptor cells and methylated in non-expressing cells from the inner nuclear layer. Presumably, these different patterns are established at some point between when the retinal cells are dividing neuroblasts and when they have undergone terminal division and begin to express cell-specific markers. The timing of the onset of this differential methylation found in two cell types originating from a common precursor is unknown. The research outlined in this exploratory proposal will provide a detailed molecular description of the high resolution the DNA methylation pattern around the transcription start site of two model PR-specific genes, Rbp3 and Rho, and how that methylation status changes during development. Genome-wide array experiments will examine tissue-specific DNA methylation patterns in the adult and developing retina, and will explore the hypothesis that retinal degeneration may be associated with changing patterns of retinal DNA methylation. Together, these studies will provide the necessary fundamental knowledge and set the stage for future mechanistic studies exploring the role of epigenetic mechanisms in retinal development and disease. PUBLIC HEALTH RELEVANCE: The ability of epigenetic mechanisms, such as DNA methylation, to initiate and maintain control of gene expression in the retina offers an entirely new frontier for exploration. Understanding the relationship between DNA methylation and tissue-specific gene expression in the developing and adult retina and exploring the role of DNA methylation in the molecular pathogenesis of retinal disease could someday have a significant impact on how we treat blinding diseases.

DESCRIPTION (provided by applicant): Retinal development, normal function, and disease are controlled, to a large extent, by the pattern of genes expressed by the cells of the retina. Despite the significant advances being made in understanding the role of epigenetics in gene regulation in other fields, little is known about the relationship between DNA methylation patterns, retinal gene expression, and retinal disease. In the United States alone, almost 4 million individuals currently have either glaucoma or age-related macular degeneration (AMD), and untreatable blindness can result despite treatment. With both diseases, gene expression changes in the retina have been observed. One modulator of gene expression is DNA methylation. We hypothesize that alterations in DNA methylation, accompany and may actually precede the gene expression changes seen with the onset of glaucoma and AMD. The proposed research will explore this hypothesis by providing a detailed, genome-wide map of the DNA methylation changes in the human retina that are associated with these two diseases. Disease-affected cell populations (retinal ganglion cells in glaucoma;photoreceptors and retinal pigment epithelium in AMD) will be individually isolated by laser capture microdissection, and compared to the same cell population from age- and sex-matched normal control eyes. Genomic DNA samples will be enriched for methylated DNA by affinity purification using immobilized MBD2-MBD. Methylated-enriched samples will be hybridized to high resolution human tiling microarray sets. Unenriched genomic DNA will be separately hybridized for calculations to adjust for probe effects. Differentially methylated areas will be identified by pair- wise comparisons between normal and diseased eyes. Nearby annotated genes will be identified as candidates for having disease-specific DNA methylation patterns and will be further investigated. The ability of epigenetic mechanisms, such as DNA methylation, to modulate patterns of gene expression in the setting of eye disease offers an entirely new frontier for exploration and has the potential to significantly affect our understanding and treatment of important ocular diseases such as glaucoma and AMD. Public Health Relevance: The ability of epigenetic mechanisms, such as DNA methylation, to initiate and modulate abnormal gene expression in the retina offers an entirely new frontier for investigation. Exploring the role of DNA methylation in the etiology, severity, and progression of glaucoma and age-related macular degeneration should lead to novel insights into the molecular mechanisms of disease pathogenesis and innovative therapeutic approaches.

DESCRIPTION (provided by applicant): Age-related macular degeneration (AMD) is a major causes of visual disability and blindness. Although significant progress has been made in identifying genetic factors that underlie AMD, the majority of factors that determine disease risk remain unknown, leading to an interest in non-genetic factors that contribute to disease. Epigenetic alterations increase over time, in part as a result of environmental factors, and may explain the late onset of common diseases like AMD. Funded by our X01 grant, CIDR has determined the genome-wide DNA methylation patterns in peripheral blood for 500 individuals including AMD and primary open-angle glaucoma (POAG) patients, as well as age- and sex-matched controls. This grant, in response to the NEI's RFA on Integrative Data Analysis, will integrate various existing datasets including genetic, epigenetic, genomic annotation, and protein-protein interactions to discover novel risk factors associated with AMD. The necessary genome data exists and is publicly available, but tools to overlay and intersect both genetic and epigenetic data are lacking. The goal of this proposal is two-fold: (1) We will develop innovative bioinformatics approaches to interpret and integrate these diverse datasets; (2) We will identify novel genetic variations, epigenetic variations and the interactions between them that are associated with AMD. We propose to design, build, and use computational tools to execute integrated epigenetic and genetic analysis to pursue the following Aims. In Aim 1, we will integrate methylation and GWAS data in novel ways to identify novel genetic variations associations with AMD at the resolution of genomic sites. The disease-specific differentially methylated regions (DMRs) will be used as an intermediary phenotype, and genetic variations associated with the DMRs will be identified. By integrating the genetic datasets obtained from the AMD GWAS studies, we will identify methylation quantitative trait loci (methQTL) that not only can provide novel genetic variations associated with diseases but also can reveal epigenetic risk factors for AMD that are mediated genetically. In Aim 2 we will identify interactions between genetic and epigenetic variations associated with AMD at the resolution of genes. We will develop a network-based approach to identify interaction modules enriched for the genes that are either genetically altered or epigenetically altered to explore how these interactions might contribute to risk of the disease. The work described in this proposal is a critical step toward a better understanding of the underlying genetic and epigenetic risk factors that contribute to the development of AMD. Our study will also provide a general computational framework for integrative analyses for other human diseases. In the future, the identified DMRs could be experimentally validated and ultimately lead to new biomarkers and therapeutic targets for AMD.