Distinct expression requirements and rescue strategies for BEST1 loss- and gain-of-function mutations

eLife

Yu Zhang¹  Yang Kong¹  Yao Li¹  Tingting Yang²  Alec Kittredge³  Yin Shen⁴  Qingqing Zhao⁵  Stephen H Tsang⁶ 

[1] Department of Ophthalmology, Vagelos College of Physicians & Surgeons, Columbia University, 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;Department of Pharmacology, Columbia University, New York, United States;Eye Center, Medical Research Institute, Renmin Hospital, Wuhan University, Wuhan, China;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;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;

关键词: bestrophin-1 (BEST1);    retinal pigment epithelium (RPE);    gene therapy;    retinal diseases;    calcium-activated chloride channel (CaCC);    gain-of-function;    Human;   

DOI  :  10.7554/eLife.67622

来源: eLife Sciences Publications, Ltd

【 摘 要 】

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.

【 授权许可】

CC BY