Mistargeting and ER retention of CLN7 patient-associated nonsense and sequence deletion mutations as a novel cause for CLN7 disease

CLN7 disease is a neurodegenerative lysosomal storage disorder caused by defects in MFSD8. We performed a comprehensive analysis of patient mutations causing CLN7 disease, variant late-infantile and non-syndromic adult phenotypes. Our analyses of protein expression and post-translational modifications, such as proteolytic cleavage and complex type N-linked oligosaccharide processing, along with double immunofluorescence analyses, demonstrated that the nonsense mutations p.Q206X, p.W456X, p.Q474X, and p.R482X, or the in-frame deletion mutation p.V109_I113del, resulted in decreased protein levels at steady state compared with wild type CLN7 and showed mistargeting and ER retention as the primary cause for loss of CLN7 function. We also investigated several missense mutations clustered in transmembrane domain 11 that affect conserved residues, which are believed to be important for CLN7 function. Analysis of protein levels, complex type N-glycosylation, proteolytic cleavage in lysosomes, and colocalization with lysosomal marker proteins in double immunofluorescence analyses showed that patient mutations p.T458L, p.R465Q, and p.R465W did not affect protein stability or correct lysosomal targeting of CLN7, indicating functional impairment. The missense mutation p.M454T resulted in increased cysteine protease-mediated turnover of mutant CLN7 in lysosomes. Using an assay to measure the generation of an enlarged endosome phenotype in cells overexpressing CLN7 carrying missense mutations, a loss of CLN7 function could not be detected. The effects of missense mutations in transmembrane domain 11 on CLN7 function remain to be investigated. In summary, our study revealed mistargeting and ER retention of nonsense and in-frame deletion mutations in MFSD8 as a cause of CLN7 disease, variant late-infantile phenotype.