Disease-causing mutations exhibit disparate effects on the localization of bestrophin-1
Mutations in BEST1 are a common cause of inherited retinal degeneration in humans. The gene encodes bestrophin-1 (Best1), a homo-oligomeric, integral membrane protein localized to the basolateral plasma membrane of the retinal pigment epithelium (RPE).
More than 200 distinct mutations have been reported to cause five distinct retinal degenerative diseases, including:
- Best vitelliform macular dystrophy (BVMD)
- Adult vitelliform macular dystrophy (AVMD)
- Autosomal recessive bestrophinopathy (ARB)
- Autosomal dominant vitreoretinochoroidopathy (ADVIRC)
- Retinitis pigmentosa (RP)
These diseases, collectively called the bestrophinopathies, are distinguished by type of inheritance, effects on the electro-oculogram and electroretinogram, age of disease onset, and location and extent of retinal lesions. The mechanisms that underlie the bestrophinopathies and determine why mutations cause one disease over another are not yet known.
Localization and oligomerization
To gain insights into these diseases, Alan D. Marmorstein, Ph.D., and a research team at Mayo Clinic's campus in Rochester, Minnesota, conducted a localization and oligomerization screen for 28 Best1 mutants associated with AVMD, ARB, ADVIRC and RP. Results of their study were published in Experimental Eye Research in 2014.
Researchers expressed the mutants, fused to yellow fluorescent protein, in polarized Maden-Darby canine kidney monolayers. The team screened the mutants for defects in localization and oligomerization using confocal microscopy and immunofluorescence, live-cell fluorescence resonance energy transfer (FRET) and reciprocal coimmunoprecipitation experiments.
"All 28 mutants exhibited comparable FRET efficiencies to and coimmunoprecipitated with wild-type (WT) Best1, indicating unimpaired oligomerization," says Dr. Marmorstein. "RP- and ADVIRC-associated mutants were properly localized to the basolateral plasma membrane of cells, while two AVMD and most ARB mutants were mislocalized. When coexpressed, all mislocalized mutants caused mislocalization of WT Best1 to intracellular compartments."
The results indicate that:
- Mislocalization of Best1 is not an absolute feature of any individual bestrophinopathy, since it occurs in BVMD, AVMD and ARB
- Mislocalization of Best1 is not a cause of disease, since some ARB mutants that do not also cause dominant disease cause mislocalization of Best1
- Absence of Best1 activity from the plasma membrane is tolerated
The research team also found that ARB truncation mutants L174Qfs*57 and R200X can form oligomers with WT Best1, indicating that the first approximately 174 amino acids of Best1 are sufficient for oligomerization to occur.
"Ultimately, this research indicates that AVMD, ARB, ADVIRC and RP are associated with disparate effects on Best1 localization, but not oligomerization," says Dr. Marmorstein. "Further research is indicated. This research did not explore specific patterns of localization, which could further distinguish individual mutations and potentially differentiate mislocalization in different diseases. The study also did not assess the functional consequences of mislocalized mutants in RPE cells."
"For now, we postulate that unique changes in calcium homeostasis, phagocytosis, binding partners or other factors may underlie the differential pathogenesis of one disease over another," says Dr. Marmorstein.
Researchers seek stem cell donors
Dr. Marmorstein's laboratory is currently using stem cells in their work to find a cure for the bestrophinopathies. Best1 is expressed only in retinal pigment epithelial cells in the eye. The most common bestrophinopathy, Best disease, is dominantly inherited.
"Stem cells from a patient with a bestrophinopathy have the same disease as the donor," says Dr. Marmorstein, "and stem cells derived from a patient's skin can be turned into retinal pigment epithelial cells in the laboratory."
Dr. Marmorstein and collaborators Jose S. Pulido, M.D., and Raymond Iezzi Jr., M.D., at Mayo Clinic, are currently recruiting patients with bestrophinopathies to provide a skin biopsy for these studies.
"We envision using patients' own stem cell-derived retinal pigment epithelial cells to determine the best course of treatment for their specific diseases," says Dr. Marmorstein. "In some cases this approach may involve gene therapy. In others, the stem cell-derived retinal pigment epithelial cells may be repaired in the laboratory and transplanted back into the patient's eye, preventing loss of vision."
For more information
Contact Dr. Marmorstein at firstname.lastname@example.org.
Development of Induced Pluripotent Stem Cells From Patients With Best Disease and Other Inherited Retinal Degenerative Diseases. Mayo Clinic Clinical Trials.
Johnson AA, et al. Disease-causing mutations associated with four bestrophinopathies exhibit disparate effects on the localization, but not the oligomerization, of bestrophin-1. Experimental Eye Research. 2014;121:74.