A Recurrent Gain-of-Function Mutation in CLCN6, Encoding the ClC-6 Cl−/H+-Exchanger, Causes Early-Onset Neurodegeneration

Abstract

Dysfunction of the endolysosomal system is often associated with neurodegenerative disease because postmitotic neurons are particularly reliant on the elimination of intracellular aggregates. Adequate function of endosomes and lysosomes requires finely tuned luminal ion homeostasis and transmembrane ion fluxes. Endolysosomal CLC Cl À /H þ exchangers function as electric shunts for proton pumping and in luminal Cl À accumulation. We now report three unrelated children with severe neurodegenerative disease, who carry the same de novo c.1658A>G (p.Tyr553Cys) mutation in CLCN6, encoding the late endosomal Cl À /H þ -exchanger ClC-6. Whereas Clcn6 À/À mice have only mild neuronal lysosomal storage abnormalities, the affected individuals displayed severe developmental delay with pronounced generalized hypotonia, respiratory insufficiency, and variable neurodegeneration and diffusion restriction in cerebral peduncles, midbrain, and/or brainstem in MRI scans. The p.Tyr553Cys amino acid substitution strongly slowed ClC-6 gating and increased current amplitudes, particularly at the acidic pH of late endosomes. Transfection of ClC-6 Tyr553Cys , but not ClC-6 WT , generated giant LAMP1-positive vacuoles that were poorly acidified. Their generation strictly required ClC-6 ion transport, as shown by transport-deficient double mutants, and depended on Cl À /H þ exchange, as revealed by combination with the uncoupling p.Glu200Ala substitution. Transfection of either ClC-6 Tyr553Cys/Glu200Ala or ClC-6 Glu200Ala generated slightly enlarged vesicles, suggesting that p.Glu200Ala, previously associated with infantile spasms and microcephaly, is also pathogenic. Bafilomycin treatment abrogated vacuole generation, indicating that H þ -driven Cl À accumulation osmotically drives vesicle enlargement. Our work establishes mutations in CLCN6 associated with neurological diseases, whose spectrum of clinical features depends on the differential impact of the allele on ClC-6 function.

The molecular and phenotypic spectrum of CLCN4‐related epilepsy

“…Remarkably, all CLCN4 variants lead to loss of function, whereas diseases associated with CLCN6 and CLCN7 can also be caused by gain-of-function mutations that increase the current amplitude or alter voltage-dependent activation kinetics. 10,[36][37][38][39] For CLCN4, it is notable that frameshift or intragenic deletion variants cause rather mild phenotypes, whereas missense variants cause more severe phenotypes. Moreover, early death occurred in four patients with missense variants, whereas it did not occur in patients with frameshift or intragenic deletion variants, which may suggest that perturbation of normal ClC-4 protein function is more deleterious than a reduction in its abundance or a complete absence.…”

“…We found two recurrent missense variants, p.Gly544Arg and p.Arg718Trp, suggesting these areas are possible mutational “hot spots” in CLCN4 . Remarkably, all CLCN4 variants lead to loss of function, whereas diseases associated with CLCN6 and CLCN7 can also be caused by gain‐of‐function mutations that increase the current amplitude or alter voltage‐dependent activation kinetics 10,36–39 . For CLCN4 , it is notable that frameshift or intragenic deletion variants cause rather mild phenotypes, whereas missense variants cause more severe phenotypes.…”

“…The CLC gene family contains nine members in mammals, four of which encode plasma membrane chloride channels (ClC-1, ClC-2, ClC-Ka, ClC-Kb) and five intracellular 2Cl − /H + exchangers (ClC-3-7). 1,2 Dysfunction of some of these leads to severe neurological disorders, including leukodystrophy, neurodegeneration, intellectual disability (ID), epilepsy, and lysosomal storage disease for ClC-2, ClC-3, ClC-4, ClC-6, and ClC-7, [3][4][5][6][7][8][9][10][11][12][13][14][15][16] highlighting the pivotal role of CLC proteins in the central nervous system. ClC-4, which is encoded by the CLCN4 (Online Mendelian Inheritance in Man database # 302910) gene located on human chromosome Xp22.2, is a voltage-dependent 2Cl − / H + exchanger.…”

From Pinocytosis to Methuosis—Fluid Consumption as a Risk Factor for Cell Death

“…Dysfunctional ClC exchangers may lead to a broad variety of symptoms and disorders including neurodegeneration and other neuropathies, proteinuria and kidney stones, osteopetrosis, albinism, and lysosomal storage diseases ( Jentsch and Pusch, 2018 ; Nicoli et al, 2019 ; Schwappach, 2020 ; Bose et al, 2021 ). Overexpression of ClC-3 or gain-of-function mutations of ClC-6 or ClC-7/Ostm1 leads to swelling of late endosomes and lysosomes ( Li et al, 2002 ; Nicoli et al, 2019 ; Polovitskaya et al, 2020 ). In B-cell non-Hodgkin lymphoma cells, the PIKFyfe inhibitor apilimod leads to CD by formation of giant vacuoles and disruption of endolysosomal function, an effect that requires functional ClC-7/Ostm1 transporters ( Gayle et al, 2017 ).…”

The Genetic Basis of Phenotypic Heterogeneity in the Neuronal Ceroid Lipofuscinoses

“…This is a slowly progressing but not life-limiting disease with no ophthalmologic abnormalities or epilepsy, and without typical ceroid/lipofuscin storage ( 29 ). A single gain of function missense mutation in CLCN6 has recently been shown to cause very severe disease in children ( 30 ) that would not be classed as NCL, although the mouse model lacking the function of the homologous gene causes mild lysosomal storage disease and the CLCN6 gene was considered a candidate gene for mild NCL disease ( 31 ).…”