title: Applying bioinformatic tools to better understand eye diseases
Creators: name: Singh, V
Publisher: name: University of Tasmania
Year: 2020

description: The highly specialized cells of the eye function in concert to produce clear vision, one of the most valued senses. Visual impairment or blindness can occur as a result of disease or trauma. Age-related macular degeneration, glaucoma, cataracts and diabetic retinopathy are common causes of vision loss in older individuals. This thesis explores the bioinformatics approaches based on the central dogma as a model for exploring the experimental models for human eye diseases including quality control of stem cells to detect chromosomal abnormalities; epigenetic age prediction of ocular tissues; identification of novel genes in Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) knockout screening in uveal melanoma; and RNA-Seq analysis to understand the molecular mechanisms in oxygen-induced retinopathy and differentiation of dental pulp mesenchymal stem cells into trabecular meshwork cells. In a virtual karyotyping study, I investigated the utility of a low-density genome-wide SNP array for the karyotypic assessment of human pluripotent stem cells (hPSCs) using the Illumina Infinium HumanCore BeadChip. Specifically, the resolution of these arrays in detecting chromosomal aberrations and their ability to identify clonal variations was determined. It was shown that the SNP array can detect chromosomal abnormalities when at least 25% of the cell population is aberrant. Our data demonstrate that an array-based karyotyping offers an economical and robust sampling method, in the genomic resolution, compared with standard cytogenetic karyotyping of hPSCs. This approach could provide a reliable, rapid and costeffective assessment of hPSCs clonality and virtual karyotype for large-scale generation and maintenance of hPSCs. To investigate the effects of aging among different tissues, we calculated the DNA methylation age of whole peripheral blood and ocular tissue from the same individual and compared it with the person’s chronological age. We found significant differences between chronological and epigenetic ages (p&lt;0.048). Our study showed that there is a significant difference (mean = 44.4 years) between chronological and epigenetic age in neurosensory retinal tissue and the same pattern identified by various tools. Through a CRISPR knockout screening study, we used the Human GeCKOv2 pooled library in OCM1 cell lines (uveal melanoma) to identify novel genes that are associated with tumorigenesis using the CRISPRAnalyzeR tool on next-generation sequencing data. Overall, from our analysis, we found 15 genes that have relatively low expression in the passage 12 as compared to the passage 0 and are associated with the metabolic process, cellular process, primary metabolic process, cellular metabolic process, biological process and organic substance metabolic process. Our study shows that these genes are crucial for cell proliferation; however, further in-vitro and in-vivo validation is required. In RNA-Seq studies, we investigated the miRNA expression in oxygen-induced retinopathy (OIR) rat models through next-generation sequencing (RNA-Seq) data. Our RNA-Seq data suggested that the expression of miR-143 dysregulated and mediated the regulatory networks, leading to retinal neovascularization. Furthermore, miR-143 can influence cellular motion and cell-matrix interaction during vascular formation. We also found that miR-126, miR-150 is significantly down-regulated and directly involved in retinal neovascularization. We also investigated the propensity of mesenchymal stem cells (MSC) populations (dental pulp-derived MSCs) to differentiate into trabecular meshwork (TM) cells. We performed RNA sequencing under two conditions: control (DPMSCs) and treated (with growth factors to differentiate into the TM cells). Overall, we found over 8000 genes that are statistically significant and have an association with tissue development, extracellular matrix development and related pathways, and demonstrated the ability of DPMSCs to differentiate into TM cells. Using bioinformatics techniques to develop models of blinding eye diseases can lead to new and better treatments to preserve vision.