Integration of eQTL and single-cell transcriptomics in the human eye identifies causal genes for age-related macular degeneration
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE135092 | GEO | 2020/01/22
REPOSITORIES: GEO
Similar Datasets
Project description:Purpose: The goals of this study were to identify cell specific expression patterns of the retinal pigment epithelium (RPE) and a variety of choroidal cell types originating from the macula and the periphery of human donor eyes, with a particular emphasis on identifying expression signatures of choroidal endothelial cells. Methods: Independent libraries were prepared for macular and peripheral samples of combined RPE/choroid from seven donors in two single cell sequencing experiments. In the first experiment, 8-mm macular and peripheral punches of RPE/choroid from Donors 1-3 were digested in papain prior to cryopreservation and subsequent GEM barcoding/library construction. Donors 1-2 had no noted ophthalmologic disease documented, while Donor 3 was diagnosed with age-related macular degeneration. In the second experiment, 12-mm macular and peripheral punches of RPE/choroid from Donors 4-7 were digested in collagenase II prior to cryopreservation, CD31-magentic bead enrichment and subsequent GEM barcoding/library construction. Donors 5-7 had no noted ophthalmologic disease documented, while Donor 4 was diagnosed with age-related macular degeneration. In both experiments, libraries were sequenced on a HiSeq4000. Sequenced reads were mapped to the human genome build hg19 will CellRanger(v3.0.1) and filters removed cells likely to be doublets or cells with a high proportion of mitochondrial reads. Clustering of cells with similar expression profiles was performed with Seurat (v3.0.2). Results: In the first experiment with unenriched RPE/choroid from donors 1-3, we recovered 4,355 cells post filtering with 40,177,477 corresponding reads. A total of 2,167 cells originated from the macula and 2,168 cells originated from the periphery. A total of 11 clusters were identified, and highly enriched genes in each cluster were used to classify clusters into their presumed dominant cell type. Differential expression analysis was performed between cells of macular and peripheral origin in each cluster and between donors with and without a history of age-related macular degeneration. Results: In the second experiment with CD31-enriched RPE/choroid from donors 4-7, we recovered 14,234 cells post filtering with 135,742,297 corresponding reads. A total of 7,647 cells originated from the macula and 6,587 cells originated from the periphery. A total of 8,521 of these cells were presumed endothelial cells. A total of 13 clusters were identified, and highly enriched genes in each cluster were used to classify clusters into their presumed dominant cell type(s). Differential expression analysis was performed between cells of macular and peripheral origin in each cluster and between donors with and without a history of age-related macular degeneration. Within each of the endothelial clusters (Clusters 5-8), differential expression analysis was performed between each cluster and the remaining endothelial cells. Conclusions: This study provides a large atlas of single-cell level gene expression patterns of the human retinal pigment epithelium and choroidal cell types. We identify expression patterns of most expected choroidal cell populations and describe macular versus peripheral regional differences. We additionally identify enriched transcripts in specific cell populations in age-related macular degeneration. We characterize gene expression patterns along the choroidal vascular tree, and identify populations of arterial, capillary, and venous endothelial cells. Our results show that single-cell sequencing can be performed on human RPE/choroid after cryopreservation, and that CD31 magnetic bead enrichment can be employed to enrich for endothelial cells for single-cell sequencing.
2019-10-30 | GSE135922 | GEO
Project description:<p>Proper spatial differentiation of retinal cell types is necessary for normal human vision. Many retinal diseases, such as Best disease and male germ cell associated kinase (MAK)-associated retinitis pigmentosa, preferentially affect distinct topographic regions of the retina. While much is known about the distribution of cell types in the retina, the distribution of molecular components across the posterior pole of the eye has not been well-studied. To investigate regional difference in molecular composition of ocular tissues, we assessed differential gene expression across the temporal, macular, and nasal retina and retinal pigment epithelium (RPE)/choroid of human eyes using RNA-Seq. RNA from temporal, macular, and nasal retina and RPE/choroid from four human donor eyes was extracted, poly-A selected, fragmented, and sequenced as 100 bp read pairs. Digital read files were mapped to the human genome and analyzed for differential expression using the Tuxedo software suite. Retina and RPE/choroid samples were clearly distinguishable at the transcriptome level. Numerous transcription factors were differentially expressed between regions of the retina and RPE/choroid. Photoreceptor-specific genes were enriched in the peripheral samples, while ganglion cell and amacrine cell genes were enriched in the macula. Within the RPE/choroid, RPE-specific genes were upregulated at the periphery while endothelium associated genes were upregulated in the macula. Consistent with previous studies, BEST1 expression was lower in macular than extramacular regions. The MAK gene was expressed at lower levels in macula than in extramacular regions, but did not exhibit a significant difference between nasal and temporal retina. The regional molecular distinction is greatest between macula and periphery and decreases between different peripheral regions within a tissue. Datasets such as these can be used to prioritize candidate genes for possible involvement in retinal diseases with regional phenotypes. </p> <p>Reprinted from <a href="https://www.ncbi.nlm.nih.gov/pubmed/?term=25446321">Whitmore, S.S., Wagner, A.H., DeLuca, A.P., Drack, A.V., Stone, E.M., Tucker, B.A., Zeng, S., Braun, T.A., Mullins, R.F., Scheetz, T.E., 2014. Transcriptomic analysis across nasal, temporal, and macular regions of human neural retina and RPE/choroid by RNA-Seq. Experimental Eye Research</a>. Used under <a href="http://creativecommons.org/licenses/by-nc-sa/3.0/">Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported </a>license.</p> <p>Additional RNA sequencing was performed on temporal, macular, nasal, inferior, and superior retina from a fifth subject and is included in this dataset. See original publication for details.</p>
| phs001151 | dbGaP
Project description:Age-related macular degeneration (AMD) is a leading cause of human blindness in developed countries. While significant inroads have been made over the past decade, an integrated description of the molecular mechanisms underlying AMD has yet to emerge. Here we describe a systems-level transcriptome analysis of the retina and retinal pigmented epithelium (RPE)-choroid complex from 31 normal, 26 AMD, and 11 potential pre-AMD human eyes derived from the University of Iowa. Our analysis identifies cell-mediated immune responses as the central feature of dry AMD, wet AMD, and geographic atrophy (GA), and we confirm this finding using a second cohort of donor eyes obtained from the Lion's Eye Bank of Oregon. In addition, in the RPE-choroid, we identify the major up-regulated pathways in GA and wet AMD as apoptosis and angiogenesis, respectively. In the retina, a graded up-regulation of wound response, complement, and neurogenesis pathway genes strongly correlates with advanced stages of AMD, in parallel with a progressive down-regulation of key phototransduction processes. Finally, using expression signatures enriched in functional pathways, we assemble two detailed AMD interactomes that highlight modular gene expression programs that delineate and interconnect dry, wet, and GA AMD subtypes across both RPE-choroid and retina tissues. In total, these interactomes are comprised of over 150 genes of which 23 have been previously associated with AMD. These data provide new insights into the expression landscape of AMD pathophysiology, and reveal numerous new targets for AMD pharmaceuticals and diagnostics.
2012-02-14 | GSE29801 | GEO
Project description:Age related macular degeneration (AMD) is the most prominent cause of blindness in the developed countries. Vision loss in the advanced stages of the disease is caused by atrophy of retinal photoreceptors, overlaying retinal pigment epithelium (RPE) and choroid endothelial cells. The molecular events that underline the development of these cell types from in utero to adult as well as the progression from the asymptomatic, to intermediate and advanced stages AMD are not yet fully understood. We performed single cell RNA-Sequencing (Seq) of human fetal and adult RPE and choroid, profiling in detail all the cell types and elucidating proliferation, differentiation, antigen pathway presentation and immunomodulation events that characterise the first half of human gestation. Our data demonstrate that progression from fetal to adult state is characterised by an increase in expression of genes involved in oxidative stress response and detoxification from heavy metals, suggesting a better defence against oxidative stress in the adult RPE and choroid tissues. Single cell comparative transcriptional analysis between an intermediate AMD patient and unaffected subject revealed specific loss of RPE and melanocytes in the macular region. While the CECs loss was not supported by our single cell RNA-Seq data, pathway enrichment analyses supported the onset of apoptosis in these cells, suggesting that during the course of AMD pathogenesis, endothelial cells are the most likely cell type to atrophy in the macula of AMD patients. Together these finds provide a rich resource for enhancing our understanding the development of RPE and choroid and the role they play in AMD progression and pathogenesis
2023-01-19 | GSE210543 | GEO
Project description:Purpose: Age-related degeneration (AMD) is a major cause of blindness in developed countries. The molecular pathogenesis of early events in AMD is poorly understood. We investigated differential gene expression in samples of human retinal pigment epithelium (RPE)/choroid from early AMD and control maculas using exon-based arrays. Methods: Gene expression levels in nine early AMD and nine control human donor eyes were assessed using Affymetrix Human Exon ST 1.0 arrays. Two controls did not pass quality control and were removed. Differentially expressed genes were annotated using DAVID, and gene set enrichment analysis (GSEA) was performed on RPE-specific and endothelium-associated gene sets. CFH genotype was also assessed and differential expression was analyzed with respect to high AMD risk (YH/HH) and low AMD risk (YY) genotypes. Results: Seventy-five genes were identified as differentially expressed (raw p-value < 0.01; >50% fold change, mean log2 expression level in AMD or control M-bM-^IM-% median of all average gene expression values); however, no genes were significant (adj. p-value < 0.01) after correction for multiple hypothesis testing. Of 52 genes with decreased expression in AMD (fold change < 0.5; raw p-value < 0.01), 18 genes were identified by DAVID analysis as associated with vision or neurological processes. GSEA of RPE-associated and endothelium-associated genes revealed a significant decrease in genes typically expressed by endothelial cells in the early AMD group compared to controls, consistent with previous histologic and proteomic studies. Analysis with respect to CFH genotype indicated decreased expression of ADAMTS9 in eyes with high-risk genotypes (fold change = -2.61; raw p-value = 0.0008). Conclusions: GSEA results suggest that RPE transcripts are preserved or elevated in early AMD, concomitant with loss of endothelial cell marker expression. These results are consistent with the notion that choroidal endothelial cell dropout occurs early in the pathogenesis of AMD. Using AltAnalyze (ver. 2.0.7 beta), we analyzed nine early AMD and nine control eyes using Affymetrix Human Exon ST 1.0 arrays. Following initial processing in AltAnalyze, two control arrays were identified as potential outliers by tests implement in arrayQualityMetrics, a package for R.
2013-12-23 | E-GEOD-50195 | biostudies-arrayexpress
Project description:We investigated the gene expression of the human TM. We isolated TM cells from healthy human donor eyes. Next, we performed RNA isolation, amplification, labeling and hybridization against 44k Agilent microarrays. We performed the microarrays against a common reference sample, namely human RPE/choroid RNA. We performed 2 replicates of human TM samples from 2 different donors.
2013-09-12 | E-GEOD-50784 | biostudies-arrayexpress
Project description:The purpose of this study was to explore diurnal gene expression changes that occur in the RPE/Choroid/Sclera. Mice C57BL/6J were purchased from the Jackson Laboratory (Bar Harbor, ME) and raised to approximately 2 months of age and entrained to a 12hr/12hr light/dark cycle for two weeks. Eye cups from male mice were rapidly dissected and RPE/ choroid/sclera tissues were collected over three consecutive diurnal cycles at Zeitgeber time (ZT) 0.5, 1, 1.5, 4, 11, 13, 16, and 23 hrs, for a total of 24 time points. Three mice were used for each time point and dissections for dark time points were done under dim-red light. To generate a reference RNA for microarray hybridization, whole eyes from equal numbers of male and female mice were collected at ZT6 and ZT7. Time: Samples were collected at the indicated Zeitgeber time points time series design
2015-08-09 | E-GEOD-40023 | biostudies-arrayexpress
Project description:This study aims to characterize myeloid cells collected from neuroretinas and RPE/choroid tissues of human donors at the age of over 70.
2024-01-07 | GSE208434 | GEO
Project description:Human retinal and RPE SAGE libraries. Profile of the genes expressed in the human peripheral retina, macula, and retinal pigment epithelium determined through serial analysis of gene expression (SAGE). Keywords: other
2001-10-03 | E-GEOD-10 | biostudies-arrayexpress
Project description:Purpose: Age-related degeneration (AMD) is a major cause of blindness in developed countries. The molecular pathogenesis of early events in AMD is poorly understood. We investigated differential gene expression in samples of human retinal pigment epithelium (RPE)/choroid from early AMD and control maculas using exon-based arrays. Methods: Gene expression levels in nine early AMD and nine control human donor eyes were assessed using Affymetrix Human Exon ST 1.0 arrays. Two controls did not pass quality control and were removed. Differentially expressed genes were annotated using DAVID, and gene set enrichment analysis (GSEA) was performed on RPE-specific and endothelium-associated gene sets. CFH genotype was also assessed and differential expression was analyzed with respect to high AMD risk (YH/HH) and low AMD risk (YY) genotypes. Results: Seventy-five genes were identified as differentially expressed (raw p-value < 0.01; >50% fold change, mean log2 expression level in AMD or control ≥ median of all average gene expression values); however, no genes were significant (adj. p-value < 0.01) after correction for multiple hypothesis testing. Of 52 genes with decreased expression in AMD (fold change < 0.5; raw p-value < 0.01), 18 genes were identified by DAVID analysis as associated with vision or neurological processes. GSEA of RPE-associated and endothelium-associated genes revealed a significant decrease in genes typically expressed by endothelial cells in the early AMD group compared to controls, consistent with previous histologic and proteomic studies. Analysis with respect to CFH genotype indicated decreased expression of ADAMTS9 in eyes with high-risk genotypes (fold change = -2.61; raw p-value = 0.0008). Conclusions: GSEA results suggest that RPE transcripts are preserved or elevated in early AMD, concomitant with loss of endothelial cell marker expression. These results are consistent with the notion that choroidal endothelial cell dropout occurs early in the pathogenesis of AMD.
2013-12-23 | GSE50195 | GEO