| Author: | Kwarteng, Regina |
| Title: | Association and functional studies for genetic variants of refractive errors and myopia |
| Advisors: | Yip, Shea Ping (HTI) Huang, Chien-ling (HTI) Yap, Maurice (SO) |
| Degree: | Ph.D. |
| Year: | 2022 |
| Subject: | Eye -- Diseases -- Genetic aspects Eye -- Refractive errors Myopia Hong Kong Polytechnic University -- Dissertations |
| Department: | Department of Health Technology and Informatics |
| Pages: | xx, 253 pages : color illustrations |
| Language: | English |
| Abstract: | Half of the global population is estimated to develop myopia by 2050, and 1.5 billion will be affected by high myopia, which is the severe form of the condition associated with several ocular pathologies including blindness. The molecular mechanisms underlying myopia onset and development are yet to be fully elucidated despite years of research to uncover genetic variants and genes associated with the condition. To date, over 200 myopia-susceptibility genes have been identified from genome-wide association studies (GWAS), candidate-gene studies and family-linkage analyses. Nonetheless, the overall contribution of these genes to and their biological impacts on myopia onset and development are yet to be explored. Evidently, more research is needed to further our knowledge of the genetic underpinnings of myopia and to extend our understanding of the biological and functional roles of genetic variants in gene regulation and myopiagenesis. Thus, functional investigation, as a follow-up step after GWAS, is crucial to identifying causal variants and their respective target genes. In this regard, a three-part research study was designed to identify genetic variants associated with high myopia in the Han Chinese population, and study the regulatory and transcriptional roles of associated variants using functional techniques and assays. In part one of the study, the technique of fine-mapping was used to identify genetic variants of the VIPR2 gene associated with high myopia in the Han Chinese population. Three genotyping assays were employed; the custom-based Illumina Infinium iSelect BeadArray, the MassARRAY iPLEX assay and the unlabelled probe melting analysis. Genotyping was conducted in three stages with a fourth stage involving imputed genotypes. Single-marker association tests and haplotype analyses unveiled unique genetic features of the associated variants of the VIPR2 gene. Two groups of variants were subsequently identified; a protective group and a high-risk group with opposing effects on myopia development. Part two of the study employed functional techniques, such as the reporter gene assay, electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP), to investigate the regulatory and transcriptional roles of associated variants of the BMP4 gene. Out of six BMP4 SNPs studied, three (rs762642, rs2071047, rs17563) showed significant allele-specific effects on transcriptional activity (all P < 0.05) by the reporter gene assay. EMSA and ChIP assays were used to identify a functional YY1 transcription factor binding site upstream of the BMP4 promoter, which demonstrated differential binding to alleles of rs2761887 in retinal nuclear extracts. The third part of the study used a high-throughput massively parallel reporter assay (MPRA) to screen for putative functional variants of refractive errors and myopia. The study conducted in two-parts investigated the functional activity of over 18,000 SNPs (including insertion-deletion) comprising lead SNPs from GWAS and SNPs in strong linkage disequilibrium. A focused screen of SNPs of VIPR2 showed previously classified high-risk and protective SNPs down-and-upregulated respectively in ocular cell types. A genome-wide screen identified over 1,000 putative functional SNPs with in ARPE-19, Muller and scleral fibroblast primary cells. In conclusion, the entirety of this thesis has explored the molecular mechanisms underlying the development of myopia by identifying genetic variants associated with high myopia and provided insight into the biological effects of sequence variation in regulation of myopia susceptibility genes. |
| Rights: | All rights reserved |
| Access: | open access |
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