Autosomal Recessive Bestrophinopathy Is Not Associated With the Loss of Bestrophin-1 Anion Channel Function in a Patient With a Novel BEST1 Mutation

摘要

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 Cla?? were assessed in transfected HEK293 cells using whole-cell patch clamp. Best1 localization was assessed by confocal microscopy in differentiated, human-induced 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, Best1I366fsX18 currents were increased while Best1R141H Cla?? currents were diminished. Coexpression of Best1R141H with Best1 or Best1I366fsX18 resulted in rescued channel activity. Overexpressed Best1, Best1R141H, and Best1I366fsX18 were all properly localized in iPSC-RPE cells; Best1R141H and Best1I366fsX18 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 Best1R141H does not cause disease, while Best1R141H together with Best1I366fsX18 causes ARB. Since both combinations generate comparable Cla?? currents, this indicates that ARB in this patient is not caused by a loss of channel activity. Moreover, Best1I366fsX18 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.