Mutations in CLCN6 as a Novel Genetic Cause of Neuronal Ceroid Lipofuscinosis in Patients and a Murine Model

Objective The aim of this study was to explore the pathogenesis of CLCN6 ‐related disease and to assess whether its Cl − /H + ‐exchange activity is crucial for the biological role of ClC‐6. Methods We performed whole‐exome sequencing on a girl with development delay, intractable epilepsy, behavioral abnormities, retinal dysfunction, progressive brain atrophy, suggestive of neuronal ceroid lipofuscinoses (NCLs). We generated and analyzed the first knock‐in mouse model of a patient variant (p.E200A) and compared it with a Clcn6 −/− mouse model. Additional functional tests were performed with heterologous expression of mutant ClC‐6. Results We identified a de novo heterozygous p.E200A variant in the proband. Expression of disease‐causing ClC‐6 E200A or ClC‐6 Y553C mutants blocked autophagic flux and activated transcription factors EB (TFEB) and E3 (TFE3), leading to autophagic vesicle and cholesterol accumulation. Such alterations were absent with a transport‐deficient ClC‐6 E267A mutant. Clcn6 E200A/+ mice developed severe neurodegeneration with typical features of NCLs. Mutant ClC‐6 E200A , but not loss of ClC‐6 in Clcn6 −/− mice, increased lysosomal biogenesis by suppressing mTORC1‐TFEB signaling, blocked autophagic flux through impairing lysosomal function, and increased apoptosis. Carbohydrate and lipid deposits accumulated in Clcn6 E200A/+ brain, while only lipid storage was found in Clcn6 −/− brain. Lysosome dysfunction, autophagy defects, and gliosis were early pathogenic events preceding neuron loss. Interpretation CLCN6 is a novel genetic cause of NCLs, highlighting the importance of considering CLCN6 mutations in the diagnostic workup for molecularly undefined forms of NCLs. Uncoupling of Cl − transport from H + countertransport in the E200A mutant has a dominant effect on the autophagic/lysosomal pathway. ANN NEUROL 2024;96:608–624