Investigating the role of BEST1 protein in Best vitelliform macular dystrophy using a novel, disease-specific human embryonic stem cell line

Abstract

Best vitelliform macular dystrophy (BVMD) is a rare form of macular dystrophy involving degeneration of the retinal pigment epithelium (RPE). It is characterised by accumulation of fluid and lipofuscin in the macula region, and consequently shares similarities with one of the leading causes of blindness, age-related macular degeneration. BVMD is known to be caused by mutations in the BEST1 gene, which encodes for the Bestrophin-1 (BEST1) protein. Over 100 BVMD-causing BEST1 mutations have been reported including the F305S mutation. The aim of this thesis was to characterise a new and unreported human embryonic stem (ES) cell line containing the F305S BEST1 mutation to better understand the molecular pathology of BVMD. The first experimental chapter focused on characterising the pluripotent properties of the F305S-mutant BEST1 human ES cell line and its capacity to differentiate into RPE. The results revealed the F305S-mutant BEST1 human ES cells were highly similar to normal human ES cells, and that RPE derived from both normal and F305S-mutant BEST1 human ES cells displayed defining RPE properties including: pigmentation, polygonal morphology and expression of important RPE genes and proteins. A key finding was that BEST1 protein in the F305S-mutant BEST1 RPE was shown to be localised predominantly within the cells compared to BEST1 in the normal RPE, which was localised predominantly at the plasma membrane. In the second experimental chapter, whole-cell patch clamp electrophysiology showed that RPE derived from the F305S-mutant BEST1 human ES cells had similar voltage-gated sodium and potassium currents compared to RPE derived from normal human ES cells. As heterologous overexpression systems suggest BEST1 protein may regulate chloride (Cl-) and calcium (Ca2+) movements, the third experimental chapter used whole-cell patch clamp to compare Cl- and Ca2+ currents in RPE derived from normal and F305S-mutant human ES cells. These data revealed reduced Cl- and Ca2+ conductance in the F305S-mutant BEST1 RPE compared to normal human ES cell-derived RPE. These data suggest that in human RPE BEST1 can i) act as a Ca2+-activated Cl- channel, and ii) regulate intracellular Ca2+. Importantly, while these data are consistent with data from heterologous overexpression systems, they are also biologically more relevant as they were obtained from human RPE expressing endogenous levels of BEST1 protein without BEST1 overexpression. Overall, the data presented in this thesis begin to define the molecular consequences of BEST1 mutation on both Ca2+ and Cl- conductance in human RPE. The data also indicate further investigation of human ES cell-derived RPE will provide new molecular insights and potentially identify different treatment options for BVMD and age-related macular degeneration.

Date of Award	2016
Original language	English