Investigation of the OCA2 gene control regions and their possible role in normal pigment variation

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2020

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Eisenberg, Micaela Tanya

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Human pigmentation phenotypes form a diverse spectrum and the genetic basis of pigmentation is complex. The oculocutaneous albinism type II (OCA2) gene has been shown to have a role in both normal and abnormal human pigmentation. Variants in the regulatory regions of the OCA2 gene have been suggested to influence its expression of the gene and by extension, contribute to the normal range of human pigment phenotypes. The promoter of the gene, previously identified, and an enhancer for OCA2, have been postulated as important determinants of variation in pigment phenotypes. The potential enhancer region occurs within intron 86 of the upstream neighbouring Hect Domain and RCC1-like Domain 2 (HERC2) gene which has no known pigment functions. This region has previously been characterised and determined to have enhancer-like properties where it is specifically active in melanocytes. Thus, it was hypothesised that normal variation in the promoter and enhancer regions of the OCA2 gene may alter transcriptional regulation of the gene and this may contribute to the range of normal human pigment phenotypes. The aim of this study was to investigate the normal variation of the OCA2 gene control region in black African individuals, which includes the associated promoter and this putative enhancer. During this project, the putative enhancer region was initially interrogated to determine if this region interacts with the OCA2 promoter, if it has enhancer-like properties and if these properties are exclusive to melanocytes. Hi-C, virtual chromatin conformation capture-on-chip and Chromatin Interaction Analysis by Paired-End Tag data were accessed to investigate possible interactions of the promoter and putative enhancer. Additionally, histone modification and chromatin conformation data from the Roadmap Epigenomics project were utilised to investigate enhancer-like properties of the putative enhancer region in melanocytes and other cell types. Following this, the normal variation within the OCA2 regulatory regions was investigated in publicly available data from the 1000 Genomes Project (KGP) and the African Genome Variation Project (AGVP). Common variation in the promoter and enhancer were identified in African populations and annotated with bioinformatics tools to determine if these normal variants are possibly functional in modulating OCA2 expression and impacting pigment variation in the normal population. The putative enhancer region was shown to interact with the OCA2 promoter and to have enhancer-like properties in melanocyte derived cancer cell lines and normal melanocytes. This iii confirms that the region has enhancer-functionality in melanocytes. A total of 287 variants were identified in the two regulatory regions combined, which included variants that were shared between the KGP and AGVP datasets. The evidence generated from the bioinformatic tools was pooled to narrow down the collection of variants to a set of 7 variants from the promoter and 10 variants from the enhancer which had the strongest evidence for being functionally significant. rs12913832 and rs7495174, from the enhancer and promoter respectively, had been previously associated with pigment phenotypes. These variants had the strongest collected evidence for functionality and modulating the expression of OCA2, which could affect the pigment phenotype of individuals. The purpose of this study was to characterise the normal variation in the regulatory regions of the OCA2 gene. There are variants in these regulatory regions that are likely to be functional and that differ in frequency between African and non-African populations. These variants may be associated with altered pigment phenotypes that are common in African ancestry populations. Since the OCA2 gene is involved in abnormal pigment disorders such as albinism, this work supports the search for causal mutations for associated disorders in the regulatory regions of the gene. An established catalogue of the normal variation has now been produced and could be used in future studies to help in identifying mutations that are truly pathogenic from those that are simply part of normal variation in the African population. This would prevent false positive associations of normal variation with albinism. Additionally, functional normal variants can be investigated to determine their contribution to normal pigment phenotypes in functional studies.

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A dissertation submitted in fulfilment of the requirements for the degree of Master of Science in Medicine to the Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, 2020

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