Elizabeth Au

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.

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.

Age-related macular degeneration (AMD) is a complex neurodegenerative disease and is the leading cause of blindness in the aging population. Early AMD is characterized by drusen in the macula and causes minimal changes in visual function. The later stages are responsible for the majority of visual impairment and blindness and can be either manifest as geographic atrophy (dry) or neovascular disease (wet). Available medicines are directed against the wet form and do not cure vision loss. Therefore, it is imperative to identify preventive and therapeutic targets. As the mechanism for AMD is unclear, we aim to interrogate the disease-affected tissue - the macular neural retina and macular retina pigment epithelium (RPE)/choroid. We investigated differentially expressed genes expression (DEG) across the clinical stages of AMD in meticulously dissected and phenotyped eyes using a standardized published protocol (Owen et al., 2019). Donor eyes (n=27) were obtained from Caucasian individuals with an age range of 60-94 and 63% were male, and tissue from the macula RPE/choroid and macula neural retina were taken from the same eye. Donor eyes were recovered within 6 hours post mortem interval time to ensure maximal preservation of RNA quality and accuracy of diagnosis. Eyes were then phenotyped by retina experts using multi modal imaging (fundus photos and SD-OCT). Utilizing DESeq2, followed PCA, Benjamini Hochberg adjustment to control for the false discovery rate, and Bonferonni correction for the number of paired comparisons: a total of 26,650 genes were expressed in the macula RPE/choroid and/or macula retina among which significant differential expression was found for 1,204 genes between neovascular AMD and normal eyes, 40 genes between intermediate AMD and normal eyes, and 1,194 genes between intermediate AMD and neovascular AMD. A comparison of intermediate AMD versus normal eyes included TCN2, PON1, IFI6, GPR123, and TIMD4 as being some of the most significant DEGs in the macula RPE/choroid. A comparison of neovascular AMD versus normal eyes included SLC1A2, SLC24A1, SCAMP5, PTPRN, and SEMA7A as being some of the most significant DEGs in the macula RPE/choroid. Top pathways of DEGs in the macular RPE/choroid identified through Ingenuity Pathway Analysis (IPA) for the comparison of intermediate AMD with normal eyes were interferon signaling and Th1 and Th2 activation, while those for the comparison of neovascular AMD with normal eyes were the phototransduction and SNARE signaling pathways. Allele-specific expression (ASE) in coding regions of previously reported AMD risk loci identified by GWAS (Fritsche et al, 2016) revealed significant ASEs for C3 rs2230199 and CFH rs1061170 in the macula RPE/choroid for normal eyes and intermediate AMD, and for CFH rs1061147 in the macula RPE/choroid for normal eyes and intermediate and neovascular AMD. An investigation of the 34 established AMD risk loci revealed that 75% of them were significantly differentially expressed between normal macular RPE/choroid and macular neural retina, with 75% of these loci showing higher expression in the RPE. Similarly, disease state differences for the GWAS loci were only found to be statistically differentially expressed in the macular RPE/choroid. Moreover, the known coding variants in the previously identified GWAS loci including, CFH , C3 , CFB , demonstrated ASE across AMD clinical stages in the macular RPE/choroid and not in the neural retina. These data at the bulk level underscore the importance of the RPE/choroid to AMD pathophysiology. While many bulk RNASeq data sets are publicly available, to the best of our knowledge this is one of the first publicly available datasets with both maculae RPE/choroid and macula neural retina from the same well phenotyped donor eye(s) where the macula is separated from the periphery. Our findings also underscore the importance of studying both macular tissue types to gain a full understanding of mechanisms leading to AMD. Our results provide insights into underlying biological mechanisms that may differentiate the disease subtypes and into the tissues affected by the disease.

The Mayan population of Guatemala is understudied within eye and vision research. Studying an observational homogenous, geographically isolated population of individuals seeking eye care may identify unique clinical, demographic, environmental and genetic risk factors for blinding eye disease that can inform targeted and effective screening strategies to achieve better and improved health care distribution. This study served to: (a) identify the ocular health needs within this population; and (b) identify any possible modifiable risk factors contributing to disease pathophysiology within this population. We conducted a cross-sectional study with 126 participants. Each participant completed a comprehensive eye examination, provided a blood sample for genetic analysis, and received a structured core baseline interview for a standardized epidemiological questionnaire at the Salama Lions Club Eye Hospital in Salama, Guatemala. Interpreters were available for translation to the patients’ native dialect, to assist participants during their visit. We performed a genome-wide association study for ocular disease association on the blood samples using Illumina’s HumanOmni2.5-8 chip to examine single nucleotide polymorphism SNPs in this population. After implementing quality control measures, we performed adjusted logistic regression analysis to determine which genetic and epidemiological factors were associated with eye disease. We found that the most prevalent eye conditions were cataracts (54.8%) followed by pseudoexfoliation syndrome (PXF) (24.6%). The population with both conditions was 22.2%. In our epidemiological analysis, we found that eye disease was significantly associated with advanced age. Cataracts were significantly more common among those living in the 10 districts with the least resources. Furthermore, having cataracts was associated with a greater likelihood of PXF after adjusting for both age and sex. In our genetic analysis, the SNP most nominally significantly associated with PXF lay within the gene KSR2 (p < 1 × 10⁻⁵). Several SNPs were associated with cataracts at genome-wide significance after adjusting for covariates (p < 5 × 10⁻⁸). About seventy five percent of the 33 cataract-associated SNPs lie within 13 genes, with the majority of genes having only one significant SNP (5 × 10⁻⁸). Using bioinformatic tools including PhenGenI, the Ensembl genome browser and literature review, these SNPs and genes have not previously been associated with PXF or cataracts, separately or in combination. This study can aid in understanding the prevalence of eye conditions in this population to better help inform public health planning and the delivery of quality, accessible, and relevant health and preventative care within Salama, Guatemala.