Biallelic CLCN2 mutations cause retinal degeneration by impairing retinal pigment epithelium phagocytosis and chloride channel function
Biallelic CLCN2 mutations cause retinal degeneration by impairing retinal pigment epithelium phagocytosis and chloride channel function
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DOI：
10.1007/s00439-023-02531-7
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发表时间：
2023-03-25
期刊：
影响因子：
5.3
通讯作者：
Zhong，Xiufeng
中科院分区：
文献类型：
--
作者：
Xu，Ping;Chen，Zhuolin;Zhong，Xiufeng
CLCN 2编码一种在人体组织中广泛表达的二孔同源二聚体氯离子通道蛋白（CLC-2）。Clcn 2和视网膜之间的关联在小鼠中已得到充分证实，因为CLC-2的功能丧失可导致小鼠视网膜病变;然而，人类中由CLCN 2突变引起的眼部表型和潜在机制仍不清楚。本研究旨在使用体外过表达系统以及患者诱导的多能干细胞（iPSC）衍生的视网膜色素上皮（RPE）细胞和视网膜类器官（RO）来定义与人类视网膜变性相关的CLCN 2变体的眼部特征并揭示其致病机制。1例携带纯合c.2257C > T（p.R753X）无义CLCN 2突变的患者随访> 6年。通过多模态影像学和功能检查对眼部特征进行全面描述。患者表现为严重的双侧视网膜变性伴感光细胞和RPE丧失。在体外，突变体CLC-2保持了正确的亚细胞定位，但与HEK 293 T细胞中的野生型CLC-2相比，通道功能降低。此外，携带CLCN 2突变的患者iPSC衍生的RPE细胞表现出ClC-2氯离子通道和外段吞噬功能障碍。值得注意的是，这些功能在使用CRISPR-Cas9系统修复CLCN 2突变后得到了拯救。然而，这种变异并没有引起患者源性RO中的显著感光细胞变性，表明功能障碍的RPE可能是双等位基因CLCN 2变异介导的视网膜病变的主要原因。这项研究首次确定了人类CLCN 2相关视网膜变性的确证性眼部特征，并揭示了与双等位基因CLCN 2变体相关的致病机制，为遗传性视网膜营养不良的病因提供了新的见解。
CLCN2encodes a two-pore homodimeric chloride channel protein (CLC-2) that is widely expressed in human tissues. The association betweenClcn2and the retina is well-established in mice, as loss-of-function of CLC-2 can cause retinopathy in mice; however, the ocular phenotypes caused byCLCN2mutations in humans and the underlying mechanisms remain unclear. The present study aimed to define the ocular features and reveal the pathogenic mechanisms ofCLCN2variants associated with retinal degeneration in humans using an in vitro overexpression system, as well as patient-induced pluripotent stem cell (iPSC)-derived retinal pigment epithelium (RPE) cells and retinal organoids (ROs). A patient carrying the homozygous c.2257C > T (p.R753X) nonsenseCLCN2mutation was followed up for > 6 years. Ocular features were comprehensively characterized with multimodality imaging and functional examination. The patient presented with severe bilateral retinal degeneration with loss of photoreceptor and RPE. In vitro,mutant CLC-2 maintained the correct subcellular localization, but with reduced channel function compared to wild-type CLC-2 in HEK293T cells. Additionally, patient iPSC-derived RPE cells carrying theCLCN2mutation exhibited dysfunctional ClC-2 chloride channels and outer segment phagocytosis. Notably, these functions were rescued following the repair of theCLCN2mutation using the CRISPR-Cas9 system. However, this variant did not cause significant photoreceptor degeneration in patient-derived ROs, indicating that dysfunctional RPE is likely the primary cause of biallelicCLCN2variant-mediated retinopathy. This study is the first to establish the confirmatory ocular features of humanCLCN2-related retinal degeneration, and reveal a pathogenic mechanism associated with biallelic CLCN2 variants, providing new insights into the cause of inherited retinal dystrophies.