Distinct expression requirements and rescue strategies for BEST1 loss- and gain-of-function mutations
Qingqing Zhao,
Yang Kong,
Alec Kittredge,
Yao Li,
Yin Shen,
Yu Zhang,
Stephen H Tsang,
Tingting Yang
Affiliations
Qingqing Zhao
Eye Center, Renmin Hospital of Wuhan University, Wuhan, China; Department of Pharmacology and Physiology, University of Rochester, School of Medicine and Dentistry, Rochester, United States
Yang Kong
Department of Ophthalmology, Vagelos College of Physicians & Surgeons, Columbia University, New York, United States
Eye Center, Medical Research Institute, Renmin Hospital, Wuhan University, Wuhan, China
Yu Zhang
Department of Ophthalmology, Vagelos College of Physicians & Surgeons, Columbia University, New York, United States
Stephen H Tsang
Jonas Children’s Vision Care, Departments of Ophthalmology and Pathology & Cell Biology, Edward S. Harkness Eye Institute, Institute of Human Nutrition and Columbia Stem Cell Initiative, New York Presbyterian Hospital/Columbia University Irving Medical Center, New York, United States
Department of Pharmacology and Physiology, University of Rochester, School of Medicine and Dentistry, Rochester, United States; Department of Ophthalmology, Vagelos College of Physicians & Surgeons, Columbia University, New York, United States
Genetic mutation of the human BEST1 gene, which encodes a Ca2+-activated Cl- channel (BEST1) predominantly expressed in retinal pigment epithelium (RPE), causes a spectrum of retinal degenerative disorders commonly known as bestrophinopathies. Previously, we showed that BEST1 plays an indispensable role in generating Ca2+-dependent Cl- currents in human RPE cells, and the deficiency of BEST1 function in patient-derived RPE is rescuable by gene augmentation (Li et al., 2017). Here, we report that BEST1 patient-derived loss-of-function and gain-of-function mutations require different mutant to wild-type (WT) molecule ratios for phenotypic manifestation, underlying their distinct epigenetic requirements in bestrophinopathy development, and suggesting that some of the previously classified autosomal dominant mutations actually behave in a dominant-negative manner. Importantly, the strong dominant effect of BEST1 gain-of-function mutations prohibits the restoration of BEST1-dependent Cl- currents in RPE cells by gene augmentation, in contrast to the efficient rescue of loss-of-function mutations via the same approach. Moreover, we demonstrate that gain-of-function mutations are rescuable by a combination of gene augmentation with CRISPR/Cas9-mediated knockdown of endogenous BEST1 expression, providing a universal treatment strategy for all bestrophinopathy patients regardless of their mutation types.
