Mutational Analysis of the Defective Protease in Classic Late-Infantile Neuronal Ceroid Lipofuscinosis, a Neurodegenerative Lysosomal Storage Disorder

Abstract

The late-infantile form of neuronal ceroid lipofuscinosis (LINCL) is a progressive and ultimately fatal neurodegenerative disease of childhood. The defective gene in this hereditary disorder, CLN2, encodes a recently identified lysosomal pepstatin-insensitive acid protease. To better understand the molecular pathology of LINCL, we conducted a genetic survey of CLN2 in 74 LINCL families. In 14 patients, CLN2 protease activities were normal and no mutations were identified, suggesting other forms of NCL. Both pathogenic alleles were identified in 57 of the other 60 LINCL families studied. In total, 24 mutations were associated with LINCL, comprising six splice-junction mutations, 11 missense mutations, 3 nonsense mutations, 3 small deletions, and 1 single-nucleotide insertion. Two mutations were particularly common: an intronic G-->C transversion in the invariant AG of a 3' splice junction, found in 38 of 115 alleles, and a C-->T transition in 32 of 115 alleles, which prematurely terminates translation at amino acid 208 of 563. An Arg-->His substitution was identified, which was associated with a late age at onset and protracted clinical phenotype, in a number of other patients originally diagnosed with juvenile NCL.

Gemfibrozil and Fenofibrate, Food and Drug Administration-approved Lipid-lowering Drugs, Up-regulate Tripeptidyl-peptidase 1 in Brain Cells via Peroxisome Proliferator-activated Receptor α

“…Recent studies suggest that some misfolded variants or misprocessed proteins may also be rescued by treatment in permissive temperatures under suitable conditions (9). There have also been reports of some mutations in TPP1 (R447H), which apparently may not have any pathogenic effect (31). Moreover, a sensitive enzyme activity assay detected residual levels of TPP1 activity in various biological samples from patients who were confirmed to have LINCL by genetic analysis (30).…”

“…Mouse Primary Astrocytes-There have been reports that residual TPP-I activity can be found in patients indicating that a few copies of normal Cln2 gene are left in patients affected with LINCL (8,9,30,31). We examined if FDA-approved lipid-lowering drugs like gemfibrozil and fenofibrate were capable of up-regulating the expression of TPP1 in brain cells.…”

AAV2-mediated CLN2 gene transfer to rodent and non-human primate brain results in long-term TPP-I expression compatible with therapy for LINCL

“…Several features of LINCL make it a good AAV-mediated CNS transfer of CLN2 D Sondhi et al candidate for gene therapy, including the monogenetic nature of the disease, knowledge of the deficient TPP-I protein and the gene (CLN2) that encodes it, a need for only low target levels of the therapeutic protein, and the ability of this protein to be secreted and internalized into neighboring cells providing potential for cross-correction. 4,21,23,30 In regard to technologies available today, gene therapy is the best option for this disorder to protect neural cells from death. The present study describes the successful delivery and long-term expression of CLN2 by an AAV2-based vector, and the assessment of its gene product, TPP-I, following delivery to rat and non-human primate brain.…”

“…Individuals who are heterozygous for LINCL have no disease phenotype suggesting that it will not be necessary to completely restore normal TPP-I levels. [1][2][3] Furthermore, individuals with LINCL who have a mutation in CLN2 that results in 5-10% of normal TPP-I activity have delayed onset of the symptoms, 23 suggesting that as little as 5% of normal activity spread throughout the brain represents a reasonable target level for clinical benefit.…”

“…17 Finally, comparisons of genotype and phenotype of children with CLN2 mutations reveal that expression as low as 5% of normal TPP-I levels lead to a much milder form of the disease with a delayed age of onset, less severe symptoms, and a prolonged lifespan, suggesting that this level of enzyme activity is a reasonable target for therapy. 23 This target level of expression of the transferred cDNA is within levels that could be achieved with current AAV vector technology.…”