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 or ClC-6 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 mutant. Clcn6 mice developed severe neurodegeneration with typical features of NCLs. Mutant ClC-6, 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 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.