Bi-allelic loss-of-function variants in CLCN2 cause retinal degeneration via impairing phagocytosis in patient retinal pigment epithelium

CLCN2 encodes a two-pore homodimeric chloride channel protein (CLC-2) widely expressed in human tissues. Previous studies reminded the retinopathy in several individuals carrying the loss-of-function variants in CLCN2, but the genotype-phenotype relationship and the underlying mechanisms remain unknown. In this study, we followed up a patient carrying the homozygous c.2257C>T (p.R753X) nonsense mutation in CLCN2 for more than 6 years, and comprehensively characterized the ocular features by multimodality imaging and functional examinations. The patient presented severe bilateral retinal degeneration with the loss of photoreceptor and retinal pigment epithelium (RPE). Moreover, the pathogenicity of this mutation was explored using an overexpression system and the patient specific induced pluripotent stem cells (iPSCs) derived RPE cells and retinal organoids (ROs). The mutant CLC-2 maintained the correct subcellular localization but displayed lower channel function than wild-type CLC-2 in HEK293T cells. Especially, the mutation also caused dysfunction of both ClC-2 chloride channel and outer segment phagocytosis in patient iPSC derived RPE cells, which were verified by RNA-seq analysis. And these impairments were rescued by repairing the CLCN2 mutation with CRISPR-Cas9 editing. However, this variant did not cause evident changes of photoreceptors in patient ROs, where RPE cells were not opposite to them as in vivo, implying the dysfunctional RPE is the primary cause of the disease. Collectively, we firstly defined the ocular features mediated by bi-allelic CLCN2 variants in detail and illustrated the pathogenetic mechanisms. Our findings will facilitate the diagnosis, treatment and prevention of CLCN2 related retinal degeneration. Overall design: Comparative gene expression profiling analysis of RNA-seq data for hiPSC-RPE cells derived from control individual and the LKPAT patient hiPSCs. HiPSC-RPE cells were collected at day 28. Total RNA from three independent biological replicates of hiPSC-RPE cells were extracted using Trizol reagent kit (T9424, Sigma). Illumina TruSeq RNA Sample Prep Kit (FC-122-1001) was used with 1 ug of total RNA for the construction of sequencing libraries. RNA libraries were prepared for sequencing using standard Illumina protocols. Illumina Casava2.19 software used for basecalling. Sequenced reads were trimmed for adaptor sequence, and masked for low-complexity or low-quality sequence, then mapped to hg38 whole genome using hisat2 v2.0.5 with parameters hisat2.Fragments Per Kilobase of exon per Megabase of library size (FPKM) were calculated using a protocol from Trapnell,Cole et. al.