Identification of 58 novel mutations in Niemann-Pick disease type C: Correlation with biochemical phenotype and importance ofPTC1-like domains inNPC1

Abstract

The two known complementation groups of Niemann-Pick Type C disease, NPC1 and NPC2, result from non-allelic protein defects. Both the NPC1 and NPC2 (HE1) gene products are intimately involved in cholesterol and glycolipid trafficking and/or transport. We describe mutation analysis on samples from 143 unrelated affected NPC patients using conformation sensitive gel electrophoresis and DNA sequencing as the primary mutation screening methods for NPC1 and NPC2, respectively. These methods are robust, sensitive, and do not require any specialized laboratory equipment. Analyses identified two NPC1 mutations for 115 (80.4%) patients, one NPC1 mutation for 10 (7.0%) patients, two NPC2 mutations for five (3.5%) patients, one NPC2 mutation for one (0.7%) patient, and no mutations for 12 (8.4%) patients. Thus, mutations were identified on 251 of 286 (88%) disease alleles, including 121 different mutations (114 in NPC1 and seven in NPC2), 58 of which are previously unreported. The most common NPC1 mutation, I1061T, was detected on 18% of NPC alleles. Other NPC1 mutations were mostly private, missense mutations located throughout the gene with clustering in the cysteine-rich luminal domain. Correlation with biochemical data suggests classification of several mutations as severe and others as moderate or variable. The region between amino acids 1038 and 1253, which shares 35% identity with Patched 1, appears to be a hot spot for mutations. Additionally, a high percentage of mutations were located at amino acids identical to the NPC1 homolog, NPC1L1. Biochemical complementation analysis of cases negative for mutations revealed a high percentage of equivocal results where the complementation group appeared to be non-NPC1 and non-NPC2. This raises the possibilities of an additional NPC complementation group(s) or non-specificity of the biochemical testing for NPC. These caveats must be considered when offering mutation testing as a clinical service.

DHPLC Analysis of patients with Nevoid Basal Cell Carcinoma Syndrome reveals novelPTCHmissense mutations in the sterol-sensing domain

“…However, the exact mechanism of signalling remains to be elucidated. In the view of the current challenges in dissecting the mechanism of this important pathway, missense mutations such as those described here become important tools for modelling signalling perturbation (Bailey et al, 2003;Hime et al, 2004;Taipale et al, 2002), and the analysis of the role of the sterol sensing domains and protein trafficking in cancer and in genetic disease such as NBCCS and Niemann-Pick Syndrome (Park et al, 2003). …”

Niemann–Pick type C 1 function requires lumenal domain residues that mediate cholesterol-dependent NPC2 binding

“…We generated NPC1 domain 2 versions of three diseasecausing mutations: V378A, R404Q, and R518Q (19). R404Q (and R404W) have been reported in several patient studies and represent mutation of a residue conserved in Niemann-Pick C1-like 1 protein; R518Q (and R518W) seem to be more prevalent among Japanese NPC patients (20). The mutant glycoproteins were produced in the same quantity as the wild-type protein from conditioned media; all contained endoglycosidase H-resistant oligosaccharides (Fig.…”

Niemann-Pick Type C Disease and Intracellular Cholesterol Trafficking

“…NPC1 may work as a lipid permease (7); however, the substrate specificity and the role of the SSD in mediating permease activity have not yet been determined. In addition to the SSD, a cysteine-rich luminal loop between TMD 8 and 9 (8) and the region between amino acids 1038 and 1253 are also important for NPC1 function (9). NPC1 protein is predominantly located within the late endosomal membrane but is also transiently associated with lysosomes and the trans-Golgi network (10).…”