Patterns of Gene Expression, Splicing, and Allele-Specific Expression Vary among Macular Tissues and Clinical Stages of Age-Related Macular Degeneration.

Age-related macular degeneration (AMD) is a leading cause of blindness, and elucidating its underlying disease mechanisms is vital to the development of appropriate therapeutics. We identified differentially expressed genes (DEGs) and differentially spliced genes (DSGs) across the clinical stages of AMD in disease-affected tissue, the macular retina pigment epithelium (RPE)/choroid and the macular neural retina within the same eye. We utilized 27 deeply phenotyped donor eyes (recovered within a 6 h postmortem interval time) from Caucasian donors (60-94 years) using a standardized published protocol. Significant findings were then validated in an independent set of well-characterized donor eyes (n = 85). There was limited overlap between DEGs and DSGs, suggesting distinct mechanisms at play in AMD pathophysiology. A greater number of previously reported AMD loci overlapped with DSGs compared to DEGs between disease states, and no DEG overlap with previously reported loci was found in the macular retina between disease states. Additionally, we explored allele-specific expression (ASE) in coding regions of previously reported AMD risk loci, uncovering a significant imbalance in C3 rs2230199 and CFH rs1061170 in the macular RPE/choroid for normal eyes and intermediate AMD (iAMD), and for CFH rs1061147 in the macular RPE/choroid for normal eyes and iAMD, and separately neovascular AMD (NEO). Only significant DEGs/DSGs from the macular RPE/choroid were found to overlap between disease states. STAT1, validated between the iAMD vs. normal comparison, and AGTPBP1, BBS5, CERKL, FGFBP2, KIFC3, RORα, and ZNF292, validated between the NEO vs. normal comparison, revealed an intricate regulatory network with transcription factors and miRNAs identifying potential upstream and downstream regulators. Findings regarding the complement genes C3 and CFH suggest that coding variants at these loci may influence AMD development via an imbalance of gene expression in a tissue-specific manner. Our study provides crucial insights into the multifaceted genomic underpinnings of AMD (i.e., tissue-specific gene expression changes, potential splice variation, and allelic imbalance), which may open new avenues for AMD diagnostics and therapies specific to iAMD and NEO.

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

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.

Heritable Risk and Protective Genetic Components of Glaucoma Medication Non-Adherence.

Glaucoma is the leading cause of irreversible blindness, affecting 76 million globally. It is characterized by irreversible damage to the optic nerve. Pharmacotherapy manages intraocular pressure (IOP) and slows disease progression. However, non-adherence to glaucoma medications remains problematic, with 41-71% of patients being non-adherent to their prescribed medication. Despite substantial investment in research, clinical effort, and patient education protocols, non-adherence remains high. Therefore, we aimed to determine if there is a substantive genetic component behind patients' glaucoma medication non-adherence. We assessed glaucoma medication non-adherence with prescription refill data from the Marshfield Clinic Healthcare System's pharmacy dispensing database. Two standard measures were calculated: the medication possession ratio (MPR) and the proportion of days covered (PDC). Non-adherence on each metric was defined as less than 80% medication coverage over 12 months. Genotyping was done using the Illumina HumanCoreExome BeadChip in addition to exome sequencing on the 230 patients (1) to calculate the heritability of glaucoma medication non-adherence and (2) to identify SNPs and/or coding variants in genes associated with medication non-adherence. Ingenuity pathway analysis (IPA) was utilized to derive biological meaning from any significant genes in aggregate. Over 12 months, 59% of patients were found to be non-adherent as measured by the MPR80, and 67% were non-adherent as measured by the PDC80. Genome-wide complex trait analysis (GCTA) suggested that 57% (MPR80) and 48% (PDC80) of glaucoma medication non-adherence could be attributed to a genetic component. Missense mutations in TTC28, KIAA1731, ADAMTS5, OR2W3, OR10A6, SAXO2, KCTD18, CHCHD6, and UPK1A were all found to be significantly associated with glaucoma medication non-adherence by whole exome sequencing after Bonferroni correction (p < 10-3) (PDC80). While missense mutations in TINAG, CHCHD6, GSTZ1, and SEMA4G were found to be significantly associated with medication non-adherence by whole exome sequencing after Bonferroni correction (p < 10-3) (MPR80). The same coding SNP in CHCHD6 which functions in Alzheimer's disease pathophysiology was significant by both measures and increased risk for glaucoma medication non-adherence by three-fold (95% CI, 1.62-5.8). Although our study was underpowered for genome-wide significance, SNP rs6474264 within ZMAT4 (p = 5.54 × 10-6) was found to be nominally significant, with a decreased risk for glaucoma medication non-adherence (OR, 0.22; 95% CI, 0.11-0.42)). IPA demonstrated significant overlap, utilizing, both standard measures including opioid signaling, drug metabolism, and synaptogenesis signaling. CREB signaling in neurons (which is associated with enhancing the baseline firing rate for the formation of long-term potentiation in nerve fibers) was shown to have protective associations. Our results suggest a substantial heritable genetic component to glaucoma medication non-adherence (47-58%). This finding is in line with genetic studies of other conditions with a psychiatric component (e.g., post-traumatic stress disorder (PTSD) or alcohol dependence). Our findings suggest both risk and protective statistically significant genes/pathways underlying glaucoma medication non-adherence for the first time. Further studies investigating more diverse populations with larger sample sizes are needed to validate these findings.