Autosomal recessive bestrophinopathy is not associated with the loss of bestrophin-1 anion channel function in a patient with a novel BEST1 mutation

PURPOSE. Mutations in BEST1, encoding bestrophin-1 (Best1), cause autosomal recessive bestrophinopathy (ARB). Encoding bestrophin-1 is a pentameric anion channel localized to the basolateral plasma membrane of the RPE. Here, we characterize the effects of the mutations R141H (CGC > CAC) and I366fsX18 (c.1098_1100+7del), identified in a patient in our practice, on Best1 trafficking, oligomerization, and channel activity. METHODS. Currents of Cl^- were assessed in transfected HEK293 cells using whole-cell patch clamp. Best1 localization was assessed by confocal microscopy in differentiated, humaninduced pluripotent stem cell-derived RPE (iPSC-RPE) cells following expression of mutants via adenovirus-mediated gene transfer. Oligomerization was evaluated by coimmunoprecipitation in iPSC-RPE and MDCK cells. RESULTS. Compared to Best1, Best1^I366fsX18 currents were increased while Best1^R141H Cl^- currents were diminished. Coexpression of Best1^R141H with Best1 or Best1I366fsX18 resulted in rescued channel activity. Overexpressed Best1, Best^1R141H, and Best1^I366fsX18 were all properly localized in iPSC-RPE cells; Best1^R141H and Best1^I366fsX18 coimmunoprecipitated with endogenous Best1 in iPSC-RPE cells and with each other in MDCK cells. CONCLUSIONS. The first 366 amino acids of Best1 are sufficient to mediate channel activity and homo-oligomerization. The combination of Best1 and Best1^R141H does not cause disease, while Best1^R141H together with Best1^I366fsX18 causes ARB. Since both combinations generate comparable Cl^- currents, this indicates that ARB in this patient is not caused by a loss of channel activity. Moreover, Best1^I366fsX18 differs from Best1 in that it lacks most of the cytosolic C-terminal domain, suggesting that the loss of this region contributes significantly to the pathogenesis of ARB in this patient.