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

Adiv A. Johnson, Lori A. Bachman, Benjamin J. Gilles, Samuel D. Cross, Kimberly E. Stelzig, Zachary T. Resch, Lihua Y Marmorstein, Jose S Pulido, Alan D Marmorstein

Research output: Contribution to journalArticle

22 Citations (Scopus)

Abstract

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<sup>-</sup> 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<sup>I366fsX18</sup> currents were increased while Best1<sup>R141H</sup> Cl<sup>-</sup> currents were diminished. Coexpression of Best1<sup>R141H</sup> with Best1 or Best1I366fsX18 resulted in rescued channel activity. Overexpressed Best1, Best<sup>1R141H</sup>, and Best1<sup>I366fsX18</sup> were all properly localized in iPSC-RPE cells; Best1<sup>R141H</sup> and Best1<sup>I366fsX18</sup> 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<sup>R141H</sup> does not cause disease, while Best1<sup>R141H</sup> together with Best1<sup>I366fsX18</sup> causes ARB. Since both combinations generate comparable Cl<sup>-</sup> currents, this indicates that ARB in this patient is not caused by a loss of channel activity. Moreover, Best1<sup>I366fsX18</sup> 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.

Original languageEnglish (US)
Pages (from-to)4619-4630
Number of pages12
JournalInvestigative Ophthalmology and Visual Science
Volume56
Issue number8
DOIs
StatePublished - 2015

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Anions
Mutation
Madin Darby Canine Kidney Cells
Pluripotent Stem Cells
HEK293 Cells
Adenoviridae
Confocal Microscopy
Cell Membrane
Amino Acids
Bestrophinopathy
Genes

Keywords

  • Best1
  • iPSC-RPE
  • Localization
  • Oligomerization

ASJC Scopus subject areas

  • Ophthalmology
  • Sensory Systems
  • Cellular and Molecular Neuroscience

Cite this

Autosomal recessive bestrophinopathy is not associated with the loss of bestrophin-1 anion channel function in a patient with a novel BEST1 mutation. / Johnson, Adiv A.; Bachman, Lori A.; Gilles, Benjamin J.; Cross, Samuel D.; Stelzig, Kimberly E.; Resch, Zachary T.; Marmorstein, Lihua Y; Pulido, Jose S; Marmorstein, Alan D.

In: Investigative Ophthalmology and Visual Science, Vol. 56, No. 8, 2015, p. 4619-4630.

Research output: Contribution to journalArticle

Johnson, Adiv A. ; Bachman, Lori A. ; Gilles, Benjamin J. ; Cross, Samuel D. ; Stelzig, Kimberly E. ; Resch, Zachary T. ; Marmorstein, Lihua Y ; Pulido, Jose S ; Marmorstein, Alan D. / Autosomal recessive bestrophinopathy is not associated with the loss of bestrophin-1 anion channel function in a patient with a novel BEST1 mutation. In: Investigative Ophthalmology and Visual Science. 2015 ; Vol. 56, No. 8. pp. 4619-4630.
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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 = "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, Best1I366fsX18 currents were increased while Best1R141H Cl- 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 Cl- 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.",
keywords = "Best1, iPSC-RPE, Localization, Oligomerization",
author = "Johnson, {Adiv A.} and Bachman, {Lori A.} and Gilles, {Benjamin J.} and Cross, {Samuel D.} and Stelzig, {Kimberly E.} and Resch, {Zachary T.} and Marmorstein, {Lihua Y} and Pulido, {Jose S} and Marmorstein, {Alan D}",
year = "2015",
doi = "10.1167/iovs.15-16910",
language = "English (US)",
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pages = "4619--4630",
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number = "8",

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TY - JOUR

T1 - Autosomal recessive bestrophinopathy is not associated with the loss of bestrophin-1 anion channel function in a patient with a novel BEST1 mutation

AU - Johnson, Adiv A.

AU - Bachman, Lori A.

AU - Gilles, Benjamin J.

AU - Cross, Samuel D.

AU - Stelzig, Kimberly E.

AU - Resch, Zachary T.

AU - Marmorstein, Lihua Y

AU - Pulido, Jose S

AU - Marmorstein, Alan D

PY - 2015

Y1 - 2015

N2 - 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, Best1I366fsX18 currents were increased while Best1R141H Cl- 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 Cl- 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.

AB - 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, Best1I366fsX18 currents were increased while Best1R141H Cl- 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 Cl- 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.

KW - Best1

KW - iPSC-RPE

KW - Localization

KW - Oligomerization

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U2 - 10.1167/iovs.15-16910

DO - 10.1167/iovs.15-16910

M3 - Article

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SP - 4619

EP - 4630

JO - Investigative Ophthalmology and Visual Science

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SN - 0146-0404

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