AMD is a disease with complex inheritance and epigenetic changes . Identification of the underlying genes has been difficult. Both genomic screen (locational) and candidate gene (functional) approaches have been used. Based on numerous genetic studies of AMD, approximately 50% of the heritability of AMD can be explained by two major loci harboring coding and non-coding variations at chromosomes 1q (CFH) and 10q (ARMS2/HTRA1) [19, 20, 21, 22]. Recently, a large GWAS highlighted new genes and pathways involved in the development of AMD, including complement activation, collagen synthesis, lipid metabolism/cholesterol transport, receptor-mediated endocytosis, endodermal cell differentiation, and extracellular matrix organization, indicating that many unknown genetic changes remain to be identified in the initiation and development of AMD . In this study, we screened for novel biomarkers by combining microarray information RPE-choroid and retina tissue samples from patients with AMD as well as peripheral blood samples by overlapping relevant datasets (GSE29801 and GSE10295) using integrated bioinformatics analysis for available microarray data. This is the first study to employ this approach.
The Hyper-LGs identified are potential biomarkers of AMD methylation microarrays for pre-clinical detection in peripheral blood. Among them, four Hyper-LGs (CKB, PPP3CA, TGFB2, and SOCS2) overlapped with risk genes in the category of “macular degeneration” in PHGKB. One study revealed that CKB was unlikely to explain the significant fraction of the risk of developing AMD in a family-based association dataset including 162 families and an independent case-control dataset of 399 cases and 159 fully evaluated controls . PPP3CA is a druggable molecule that inactivates MAP3K5, but has not been widely investigated for its role in AMD. One previous study revealed AMD-related sequence variants in genes encoding PPP3CA, underlying its relationship with AMD . TGFB2 induces RPE cell and collagen gel contraction. Subretinal fibrosis contributes to the loss of vision associated with AMD, and RPE cells play a key role in the fibrotic reaction . Under hypoxia conditions, RPE cells can increase the secretion of TGFB2 and induce epithelial-mesenchymal transition, resulting in the formation of scar-like fibrous tissue in AMD . Targeted inhibition of TGFB signaling may be an effective approach for retarding AMD progression . SOCS proteins are modulators of cytokine and growth factor signaling whose aberrant regulation has been linked to a variety of inflammatory and neoplastic diseases . In a GWAS study of 919 patients with exudative AMD treated with intravitreal ranibizumab, SOCS2 was a candidate gene whose levels were associated with visual loss at month three . These results provide insight into AMD pathogenesis, but must be confirmed by in vivo and in vitro experiments. The methylation patterns of PPP3CA, TGFB2, and SOCS2 in AMD have not been previously described. We found that these genes were hypermethylated and low regulated, suggesting that aberrant methylation of these genes affects the pathogenesis of AMD. No Hypo-HGs overlapped in PHGKB, likely because of the limited number of genes identified.
Among the top 5 pathways identified in KEGG and GO, calcium signaling [30, 31], sphingolipid metabolism [32, 33], fibroblast migration [34, 35], membrane , coenzyme [37, 38, 39], and DNA binding  have been investigated in AMD. Calcium signaling, sphingolipid metabolism, and coenzyme showed strong relationships with AMD, whereas the other categories require further evaluation. Calcium signaling play a vital role in RPE cell function. Intracellular calcium mobilization activates gene expression and the secretion of inflammatory cytokines such as interleukin-8 in human RPE cells . Complement attack on RPE cells leading to cell death was also modulated by extracellular calcium and intracellular signaling mechanisms . Sphingosine 1-phosphate is a potent lipid mediator that modulates inflammatory responses and proangiogenic factors, and it has been suggested that this protein upregulates CNV and is deeply involved in the pathogenesis of exudative AMD . Free radicals play a pathogenic role in AMD, whereas coenzyme Q10 has a protective effect . A combination of acetyl-L-carnitine, n-3 fatty acids, and coenzyme Q10 benefited visual functions in early AMD . However, drug metabolism pathways such as amphetamine addiction and morphine addiction may have been identified by chance and may not be related to AMD. The specific manner in which the other pathways affect AMD development and progression must be further investigated.
In the PPI network, 23 nodes and 2 edges were established from the Hypo-HGs and 151 nodes and 73 edges were established from the Hyper-LGs. DHX9, MAPT, and PAX6 were identified as hub genes. Two core modules for Hyper-LGs were structured, including module1: HNRNPA3, DHX9, SRSF11, and SLU7 and module2: SOX1, PAX6, and DLX2. Among the hub genes and core modules above, PAX6 is expressed in retinal progenitor cells throughout retinogenesis . PAX6 is a novel regulatory gene between RPE transcription factors that controls the timing of RPE differentiation and adjacent choroid maturation, suggesting that PAX6 is involved in choroid development in the pathogenesis of AMD . Other genes have not been previously investigated in AMD.
There were some limitations to this study. First, we focused on Hyper-LGs and Hypo-HGs without analyzing contra-regulated genes; thus, further analysis is required to evaluate these genes. Second, our study was limited to only 2 datasets, and we did not conduct validation in animals or patients’ samples. Thus, the results are preliminary and larger sample sizes as well as further fundamental experiments are needed to confirm these results. Third, the clinical characteristics of AMD patients included were not analyzed because these data were not available, so the results should be conservatively interpreted.