A systems biology approach uncovers novel disease mechanisms in age-related macular degeneration

Abstract

Age-related macular degeneration (AMD) is a leading cause of blindness, affecting 200 million people worldwide. To identify genes that could be targeted for treatment, we created a molecular atlas at different stages of AMD. Our resource is comprised of RNA sequencing (RNA-seq) and DNA methylation microarrays from bulk macular retinal pigment epithelium (RPE)/choroid of clinically phenotyped normal and AMD donor eyes (n = 85), single-nucleus RNA-seq (164,399 cells), and single-nucleus assay for transposase-accessible chromatin (ATAC)-seq (125,822 cells) from the retina, RPE, and choroid of 6 AMD and 7 control donors. We identified 23 genome-wide significant loci differentially methylated in AMD, over 1,000 differentially expressed genes across different disease stages, and an AMD Müller state distinct from normal or gliosis. Chromatin accessibility peaks in genome-wide association study (GWAS) loci revealed putative causal genes for AMD, including HTRA1 and C6orf223. Our systems biology approach uncovered molecular mechanisms underlying AMD, including regulators of WNT signaling, FRZB and TLE2, as mechanistic players in disease.

Keywords: AMD; DNA methylation; GA; Muller glia; RNA-seq; age-related macular degeneration; epigenomics; geographic atrophy; rare variant genetics; retinal pigment epithelium; single-cell ATAC-seq; single-cell RNA-seq.

Graphical abstract

Figure 1

Characterization of bulk macular RPE/choroid tissues (A) Schematic representation of phenotyped bulk eye tissue analyses. Created with Biorender.com. (B) Normalized HTRA1 gene expression in bulk RNA-seq. ∗FDR < 0.05, ∗∗FDR < 0.05, and fold change > 1.5; see Table S1 (ST1B). Boxplot is drawn from 25th to 75th percentiles, horizontal bars are medians, and whiskers show ranges. (C and D) Heatmap of top DEGs in bulk RNA-seq from macular RPE/choroid for (C) pairwise comparisons of normal vs. each AMD stage, and (D) linear analysis of normal and dry AMD. (E) DMPs between normal and GA. Genes <100 kb from the cytosine are in black, genes located 100 kB to 1 Mb away are in gray, and genes labeled in red are published AMD GWAS candidates. (F and G) Venn diagrams of DEGs for (F) normal vs. GA and normal vs. NEO and (G) normal vs. eAMD, normal vs. iAMD, and normal vs. GA. See Table S1 (ST1B). (H) Venn diagram of macular RPE/choroid DEGs in normal vs. GA, DEGs in normal vs. iAMD, and closest genes to DMP in normal vs. GA. See also Figure S1.

Figure 2

Single-nucleus RNA-seq of control and AMD donor eyes (A) Overview of single-cell genomics workflow from human donor eyes. (B) Dotplot of selected marker genes for major cell types. The color intensity is the normalized average expression, and the dot size represents the percentage of nuclei in each cell type with non-zero expression of that gene. (C–E) Uniform manifold approximation and projection (UMAP) dimensionality reductions of expression in (C) major cell types, (D) non-neuronal cell types, and (E) bipolar cell subtypes. See also Figure S2.

Figure 3

Pseudo-bulk differential expression from single-nucleus RNA-seq (A–D) Dotplots showing marker gene expression in single-nucleus RNA-seq (sNuc-seq). The color intensity represents the Z score of gene expression, and the dot size represents the percentage of nuclei in each group with non-zero expression. Genes are from pseudo-bulk DE in (A) Müller glia, (B) genes indicating Müller gliosis, (C) RPE, and (D) fibroblasts. (E and F) Examples of pseudo-bulk expression per cell type per donor are in (E) Müller and (F) RPE. (G and H) RNAscope ISH for (G) CRYAB and (H) CLU. Representative images from macular sections of healthy controls (top panels) and GA (middle and bottom panels) from 3 donors each. Nearby sections were stained in (G) and (H), and the images were chosen for close vicinity in each donor. GA images are from the lesion, with lesion centers oriented to the right and the borders to the left. Dashed lines indicate removal of extra white space between the RPE and neural retina that was caused by artifactual postmortem retinal detachment. See also Figure S3.

Figure 4

Single-nucleus ATAC sequencing of control and AMD donor eyes (A–C) UMAP dimensionality reductions of chromatin accessibility in (A) major cell types, (B) non-neuronal cell types, and (C) bipolar cell subtypes. (D) Genome tracks showing chromatin accessibility for DOK5, a marker gene for bipolar subtype DB5. x axis: the genomic position; y axis: normalized sequencing counts. (E and F) UMAP of major cell types, colored by (E) ATAC accessibility of marker genes, or (F) expression of marker genes based on integration of sNuc-seq and single-nucleotide ATAC sequencing (snATAC-seq). (G) Relationship of ocular cell types to genetic risk of AMD. UMAP of chromatin accessibility for major cell types, colored by the SCAVENGE trait relevance score. (H) Genome tracks showing accessibility at the ARMS2-HTRA1 locus. The triangle marks the position of the peak overlapping the lead SNP rs3750846. Correlations between peaks and gene expression are shown as arcs connecting the peak and the transcription start site of HTRA1, and the arc color denotes the Pearson correlation R. See also Figure S4.

Figure 5

Integration of ocular data in AMD (A) Normalized KIT gene expression in bulk RNA-seq from RPE/choroid. ∗∗FDR < 0.05 and fold change > 1.5. Boxplot is drawn from 25th to 75th percentiles, horizontal bars are medians, and whiskers show ranges. (B) Violin plot showing normalized KIT expression across major cell types in sNuc-seq. (C) Genome tracks showing accessibility of KIT. (D) Immunohistochemistry staining for c-KIT/CD117. Representative images from control macula (left panel and insets 1 and 2), periphery (middle panel and insets 3 and 4), and macular sections of GA lesion borders (right panel). Choroidal melanocytes are CD117+ pigmented cells (insets 1 and 3), and mast cells are CD117+ cells with no pigmentation (insets 2 and 4). Dashed lines indicate removal of extra white space between the RPE and neural retina that was caused by artifactual postmortem retinal detachment. (E and F) Normalized gene expression in bulk RNA-seq from RPE/choroid for (E) FRZB and (F) TLE2. ∗∗FDR < 0.05 and fold change > 1.5. Boxplots are drawn from 25th to 75th percentiles, horizontal bars are medians, and whiskers show ranges. (G) Dotplot showing expression in sNuc-seq for WNT pathway genes enriched in RPE. (H) Genome tracks showing accessibility of FRZB. Correlations between peaks and gene expression are shown as arcs connecting the peak and the transcription start site of FRZB. R, Pearson correlation coefficient. See also Figure S5.