Abstract
BEST1 is a Ca2+-activated Cl− channel predominantly expressed in retinal pigment epithelium (RPE), and over 250 genetic mutations in the BEST1 gene have been identified to cause retinal degenerative disorders generally known as bestrophinopathies. As most BEST1 mutations are autosomal dominant, it is of great biomedical interest to determine their disease-causing mechanisms and the therapeutic potential of gene therapy. Here, we characterized six Best vitelliform macular dystrophy (BVMD)-associated BEST1 dominant mutations by documenting the patients’ phenotypes, examining the subcellular localization of endogenous BEST1 and surface Ca2+-dependent Cl− currents in patient-derived RPEs, and analyzing the functional influences of these mutations on BEST1 in HEK293 cells. We found that all six mutations are loss-of-function with different levels and types of deficiencies, and further demonstrated the restoration of Ca2+-dependent Cl− currents in patient-derived RPE cells by WT BEST1 gene supplementation. Importantly, BEST1 dominant and recessive mutations are both rescuable at a similar efficacy by gene augmentation via adeno-associated virus (AAV), providing a proof-of-concept for curing the vast majority of bestrophinopathies.
Highlights
Genetic mutation of the human BEST1 gene causes bestrophinopathies, which consist of a spectrum of retinal degeneration disorders including Best vitelliform macular dystrophy (BVMD)[1,2], autosomal recessive bestrophinopathy (ARB)[3], adult-onset vitelliform dystrophy (AVMD)[4,5], autosomal dominant vitreoretinochoroidopathy (ADVIRC)[6], and retinitis pigmentosa (RP)[7]
Our results illustrate the physiological influence of these six dominant mutations on retinal pigment epithelium (RPE) surface Ca2+-dependent Cl− current and the BEST1 channel function, provide structural www.nature.com/scientificreports insights into their disease-causing mechanisms, and demonstrate the rescue of BEST1 function in induced pluripotent stem cells (iPSCs)-RPE via gene supplementation
The diminished Ca2+-dependent Cl− currents in the R218H, L234P and A243T patient-derived iPSC-RPEs are in accord with the deficient Ca2+-stimulated Cl− secretion shown by Cl− sensitive fluorescent dyes in these cells[29]
Summary
Genetic mutation of the human BEST1 gene causes bestrophinopathies, which consist of a spectrum of retinal degeneration disorders including Best vitelliform macular dystrophy (BVMD)[1,2], autosomal recessive bestrophinopathy (ARB)[3], adult-onset vitelliform dystrophy (AVMD)[4,5], autosomal dominant vitreoretinochoroidopathy (ADVIRC)[6], and retinitis pigmentosa (RP)[7]. Ca2+-dependent Cl− current has been suggested to generate a critical visual response upon light exposure, namely light peak (LP)[11,12,13], which is defective in almost all BEST1-mutated patients as shown by electrooculography (EOG)[14,15] This BEST1- Cl− current- LP correlation suggests gene supplementation as a promising approach for curing bestrophinopathies. Defective Ca2+-dependent Cl− currents in patient-derived RPE cells were restored by virus-mediated supplementation of the WT BEST1 gene in a time- and dose-dependent manner. Both dominant and recessive mutations of BEST1 are rescuable at a similar efficacy, and both BV and AAV can be used as the vector for gene delivery. Our findings underscore the great potential of gene augmentation therapy in treating bestrophinopathies, including those caused by BEST1 dominant mutations
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