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Matthew Miller, Jessica Fick, Jeremy M Hoffmann, Jessica A Penticoff, Robert F Mullins, Edwin M Stone, Budd Tucker; North Carolina Macular Dystrophy: Causes and Consequences of Gene Dysregulation. Invest. Ophthalmol. Vis. Sci. 2019;60(9):2862. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
North Carolina Macular Dystrophy (NCMD) is a highly-penetrant autosomal-dominant disease characterized by macular-specific dysgenesis, accumulation of drusen-like deposits, and pigment irregularity at the level of the retinal pigment epithelium (RPE). Three mutations associated with NCMD (V1-V3) lie within a putative regulatory element upstream of the PRDM13 transcription start site. The purpose of this study was to evaluate the impact of the V1-V3 variants on gene regulation, and to characterize the downstream effects on cellular function.
In order to interrogate the tissue-specificity of regulatory activity at the NCMD-associated locus, we used DNase-seq data from over 125 individual cell types publicly available through The Encyclopedia of DNA Elements (ENCODE). Accessible chromatin was also measured experimentally using formaldehyde assisted isolation of regulatory elements (FAIRE-qPCR) in induced pluripotent stem cells (iPSC), RPE, retinal progenitor cells, and choroidal endothelial cells. We also performed in-silico analysis of DNA binding motifs to predict how the V1-V3 variants alter transcription factor binding affinity. Effects on nearby gene expression was measured by RT-qPCR in NCMD-patient derived fibroblasts and in cell-lines harboring CRISPR-Cas9 generated deletions. Furthermore, in order to understand the functional effects of PRDM13 mis-expression, we created cell lines either over-expressing PRDM13 or with the gene knocked-out.
Among tissues of the eye, we found that chromatin accessibility at the NCMD-associated locus was specific to the RPE, iris pigment epithelium, and conjunctival fibroblasts. The NCMD-associated sequence variants within V1-V3 are predicted to decrease the relative binding affinity for several repressive factors, including EHF and RUNX1, and increase affinity for several activating factors, including OCT2 and MYC.
The non-coding variants that cause NCMD disrupt the function of a regulatory element that is active in RPE. Elucidation of the underlying genetic, molecular, and cellular causes of NCMD will provide insight into the epigenetic regulation of pathways that contribute to the development and homeostasis of the human macula.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
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