Niemann-Pick C1 disease: correlations between NPC1 mutations, levels of NPC1 protein, and phenotypes emphasize the functional significance of the putative sterol-sensing domain and of the cysteine-rich luminal loop

Abstract

To obtain more information of the functional domains of the NPC1 protein, the mutational spectrum and the level of immunoreactive protein were investigated in skin fibroblasts from 30 unrelated patients with Niemann-Pick C1 disease. Nine of them were characterized by mild alterations of cellular cholesterol transport (the "variant" biochemical phenotype). The mutations showed a wide distribution to nearly all NPC1 domains, with a cluster (11/32) in a conserved NPC1 cysteine-rich luminal loop. Homozygous mutations in 14 patients and a phenotypically defined allele, combined with a new mutation, in a further 10 patients allowed genotype/phenotype correlations. Premature-termination-codon mutations, the three missense mutations in the sterol-sensing domain (SSD), and A1054T in the cysteine-rich luminal loop all occurred in patients with infantile neurological onset and "classic" (severe) cholesterol-trafficking alterations. By western blot, NPC1 protein was undetectable in the SSD missense mutations studied (L724P and Q775P) and essentially was absent in the A1054T missense allele. Our results thus enhance the functional significance of the SSD and demonstrate a correlation between the absence of NPC1 protein and the most severe neurological form. In the remaining missense mutations studied, corresponding to other disease presentations (including two adults with nonneurological disease), NPC1 protein was present in significant amounts of normal size, without clear-cut correlation with either the clinical phenotype or the "classic"/"variant" biochemical phenotype. Missense mutations in the cysteine-rich luminal loop resulted in a wide array of clinical and biochemical phenotypes. Remarkably, all five mutant alleles (I943M, V950M, G986S, G992R, and the recurrent P1007A) definitively correlated with the "variant" phenotype clustered within this loop, providing new insight on the functional complexity of the latter domain.

Figure 1

Topology of the mutations identified in 30 patients on an NPC1 protein model and genotype/phenotype correlations. The schematic NPC1 protein model is drawn as proposed by Davis and Ioannou (2000). The hatched areas indicate the putative transmembrane domains. The dotted oval frame delimits the SSD. →, Frameshift mutations, all of them observed in patients with a severe infantile neurological form. ▪, Missense or nonsense mutation definitively associated to a severe infantile neurological form. □, missense mutation likely correlated with a severe infantile neurological form. ▴, Missense mutation definitively correlated with a variant biochemical phenotype. ●, Other missense mutation.

Figure 2

Western blot of NPC1 protein in cultured fibroblasts from control subjects, 12 patients with NPC1, and 1 patient with NPC2. A C-terminal affinity-purified polyclonal antibody was used. Cells from a patient with a 845delT frameshift mutation served as a negative control. Eight patients with NPC1 had a homozygous missense mutation (lanes with a single label), and three were compound heterozygotes (lanes with a dual label). The NPC2 cell line carried a homozygous E20X mutation of the HE1 gene.