title: Autosomal Recessive Bestrophinopathy Is Not Associated With the Loss of Bestrophin-1 Anion Channel Function in a Patient With a Novel BEST1 Mutation.
abstract: OBJECTIVE
  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, 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, Best1 I366fsX18 currents were increased while Best1 R141H Cl- currents were diminished. Coexpression of Best1 R141H with Best1 or Best1 I366fsX18 resulted in rescued channel activity. Overexpressed Best1, Best1 R141H, 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.
authors.affiliation: Department of Ophthalmology Mayo Clinic, Rochester, Minnesota, United States.
authors.forename: Adiv A
authors.initials: AA
authors.lastname: Johnson
authors.affiliation: Department of Ophthalmology Mayo Clinic, Rochester, Minnesota, United States.
authors.forename: Lori A
authors.initials: LA
authors.lastname: Bachman
authors.affiliation: Department of Ophthalmology Mayo Clinic, Rochester, Minnesota, United States.
authors.forename: Benjamin J
authors.initials: BJ
authors.lastname: Gilles
authors.affiliation: Department of Ophthalmology Mayo Clinic, Rochester, Minnesota, United States.
authors.forename: Samuel D
authors.initials: SD
authors.lastname: Cross
authors.affiliation: Center for Regenerative Medicine, Mayo Clinic, Rochester, Minnesota, United States.
authors.forename: Kimberly E
authors.initials: KE
authors.lastname: Stelzig
authors.affiliation: Center for Regenerative Medicine, Mayo Clinic, Rochester, Minnesota, United States.
authors.forename: Zachary T
authors.initials: ZT
authors.lastname: Resch
authors.affiliation: Department of Ophthalmology Mayo Clinic, Rochester, Minnesota, United States.
authors.forename: Lihua Y
authors.initials: LY
authors.lastname: Marmorstein
authors.affiliation: Department of Ophthalmology Mayo Clinic, Rochester, Minnesota, United States 3Department of Molecular Medicine, Mayo Clinic, Rochester, Minnesota, United States.
authors.forename: Jose S
authors.initials: JS
authors.lastname: Pulido
authors.affiliation: Department of Ophthalmology Mayo Clinic, Rochester, Minnesota, United States.
authors.forename: Alan D
authors.initials: AD
authors.lastname: Marmorstein
mesh_terms: D000293:Adolescent; D000075002:Bestrophins; D015153:Blotting, Western; D002462:Cell Membrane / Q000378:metabolism; D018118:Chloride Channels / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D004247:DNA / Q000235:genetics*; D004252:DNA Mutational Analysis; D015785:Eye Diseases, Hereditary / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D005136:Eye Proteins / Q000096:biosynthesis / Q000235:genetics*; D005260:Female; D005451:Fluorescein Angiography; D005654:Fundus Oculi; D005786:Gene Expression Regulation; D005808:Genes, Recessive; D057809:HEK293 Cells / Q000378:metabolism / Q000648:ultrastructure; D006801:Humans; D018613:Microscopy, Confocal; D008854:Microscopy, Electron; D008856:Microscopy, Fluorescence; D009154:Mutation; D018408:Patch-Clamp Techniques; D012164:Retinal Diseases / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D055213:Retinal Pigment Epithelium / Q000378:metabolism / Q000648:ultrastructure*
chemical_list: C113133:BEST1 protein, human; D000075002:Bestrophins; D018118:Chloride Channels; D005136:Eye Proteins; D004247:DNA