Patient-specific mutations impair BESTROPHIN1’s essential role in mediating Ca2+-dependent Cl- currents in human RPE

Mutations in the human BEST1 gene lead to retinal degenerative diseases displaying progressive vision loss and even blindness. BESTROPHIN1, encoded by BEST1, is predominantly expressed in retinal pigment epithelium (RPE), but its physiological role has been a mystery for the last two decades. Using a patient-specific iPSC-based disease model and interdisciplinary approaches, we comprehensively analyzed two distinct BEST1 patient mutations, and discovered mechanistic correlations between patient clinical phenotypes, electrophysiology in their RPEs, and the structure and function of BESTROPHIN1 mutant channels. Our results revealed that the disease-causing mechanism of BEST1 mutations is centered on the indispensable role of BESTROPHIN1 in mediating the long speculated Ca2+-dependent Cl- current in RPE, and demonstrate that the pathological potential of BEST1 mutations can be evaluated and predicted with our iPSC-based ‘disease-in-a-dish’ approach. Moreover, we demonstrated that patient RPE is rescuable with viral gene supplementation, providing a proof-of-concept for curing BEST1-associated diseases.

eLife digest Mutations to the gene that encodes a protein called BESTROPHIN1 cause a number of human diseases that lead to a progressive loss of sight and even blindness. Over two hundred of these disease-causing mutations exist, but it is not understood how they affect BESTROPHIN1. Furthermore, there are currently no treatments available to treat these diseases. BESTROPHIN1 is an ion channel found in cell membranes in the retinal pigment epithelium (RPE), a layer of cells in the eye that is vital for vision. When BESTROPHIN1 is stimulated by calcium ions, it opens up to allow chloride ions to flow into and out of the cell. The health of human eyes can be assessed by measuring how well they respond to light – a response that is believed to be generated from the flow of calcium-stimulated chloride ions in the RPE. Patients with mutant BESTROPHIN1 channels have an abnormally low response to light, but it remains unclear whether these channels are responsible for maintaining the flow of chloride ions required for the light response. Indeed, it is not confirmed whether calcium-stimulated chloride flow occurs on the surface of normal human RPE cells at all. Human RPE cells are difficult to obtain. Instead, Li, Zhang et al. took human skin cells – some from patients who had disease-causing mutations that affect BESTROPHIN1 – and used stem cell technology to coax the cells to develop into RPE cells. Calcium-stimulated chloride ion flow could be recorded on the surface of these cells. Next, the impact of two disease-causing mutations on BESTROPHIN1 was examined. The mutation from the patient who displayed the more severe illness completely inactivated the channel, while the other associated with milder illness caused a partial loss of channel activity. Notably, introducing normal BESTROPHIN1 into the RPE cells developed from patients with mutant BESTRPOPHIN1 restored chloride ion flow to normal levels. Thus it appears that BESTROPHIN1 is essential for maintaining calcium-stimulated chloride ion flow in human RPE cells. The techniques developed by Li, Zhang et al. form a patient-specific ‘disease-in-a-dish’ approach that could be used to study the consequences of other mutations to the gene that produces BESTROPHIN1. This work also suggests that gene therapy could potentially help to treat BESTROPHIN1-related diseases.