Investigating the Function of BEST1 in iPS-RPE Cells Derived from a Family with Autosomal Recessive Bestrophinopathy

Abstract

Purpose: Autosomal recessive bestrophinopathy (ARB) is a clinically distinct bestrophinopathy associated with slow loss of central vision and retinal pigment epithelial (RPE) cell abnormalities. ARB is a recessive disease caused by mutations in the RPE protein Bestrophin1 (BEST1). BEST1 has been proposed to act as an ion channel, however it’s exact function in the RPE has yet to be elucidated. We have produced induced pluripotent stem cells (iPSCs) from an ARB patient with a p.R200X BEST1 mutation and differentiated iPSCs into RPE in order to investigate the function of BEST1 in human RPE cells. Full length BEST1 reportedly regulates the L-type voltage dependent Ca²⁺ channels (Ca_v1.3) by decreasing the maximal currents. We investigated how the p.R200X BEST1 mutation affects the Ca_v1.3 channel functionality.

Methods: Fibroblast cells were cultured from a skin biopsy obtained from an ARB patient with a p.R200X BEST1 mutation. Fibroblasts were reprogrammed into iPSCs using episomal vectors, and pluripotency was confirmed. ARB iPSC-RPE cells were differentiated towards an RPE lineage by removal of bFGF from the culture medium. Pigmented foci were isolated and seeded to form monolayers, which were analysed by PCR, immunocytochemistry and Western blot. Ba²⁺ currents through voltage dependent Ca²⁺ channels were analysed by patch clamp technique from both ARB iPSC-RPE and human fetal iPSC-RPE.

Results: Pluripotency of patient derived iPSCs was confirmed and pigmented foci appeared in differentiated cultures after approximately 6 weeks. Purified cells formed a pigmented monolayer with classic RPE cobblestone morphology. The p.R200X mutation was confirmed in fibroblast, iPSC and RPE cells. ARB iPSC-RPE expressed classic RPE markers at the protein and RNA level, however, full length BEST1 protein was undetectable. Patch clamp recordings revealed decreased currents through Ca_v1.3 channels in ARB iPSC-RPE compared to fetal iPSC-RPE.

Conclusions: The ARB p.R200X mutation results in lack of full length BEST1 protein in RPE cells. This disrupts the interaction between BEST1 and L-type Ca²⁺ channels and reduces the currents through Ca_v1.3 channels, possibly by decreasing the number of these channels in the cell membrane.