Biomedical subjects

Gilles Millat

Publications and source records attributed to Gilles Millat.

5 recordsLinked to original sources

PubMed2006

The adult form of Niemann-Pick disease type C.

Niemann-Pick disease type C (NPC) is a fatal neurovisceral lipid storage disease of autosomal inheritance resulting from mutations in either the NPC1 (95% of families) or NPC2 gene. The encoded proteins appear to be involved in lysosomal/late endosomal transport of cholesterol, glycolipids and other molecules but their exact function is still unknown. The clinical spectrum of the disease ranges from a neonatal rapidly fatal disorder to an adult-onset chronic neurodegenerative disease. Based upon a comprehensive study of 13 unrelated adult patients diagnosed in France over the past 20 years as well as the analysis of the 55 other cases published since 1969, we have attempted to delineate the major clinical, radiological, biochemical and genotypic characteristics of adult NPC. Overall, mean age at onset (+/-SD) of neuropsychiatric symptoms was 25 +/- 9.7 years. The diagnosis of NPC was established after a mean delay of 6.2 +/- 6.4 years and the mean age at death (calculated from 20 cases) was 38 +/- 10.2 years. Major clinical features included cerebellar ataxia (76%), vertical supranuclear ophthalmoplegia (VSO, 75%), dysarthria, (63%), cognitive troubles (61%), movement disorders (58%), splenomegaly (54%), psychiatric disorders (45%) and dysphagia (37%). Less frequent signs were epilepsy and cataplexy. During the course of the disease, clinical features could be subdivided into (i) visceral signs (hepatomegaly or splenomegaly), (ii) cortical signs (psychiatric cognitive disorders and epilepsy); and (iii) deep brain signs (VSO, ataxia, movement disorders, dysarthria, dysphagia, cataplexy) which exhibited different evolution patterns. Asymptomatic and non-evolutive visceral signs were often noticed since early childhood (38.5% of our patients), followed by mild cortical signs in childhood (learning difficulties) and early adulthood (62% of cases among which 38% were psychiatric disorders). Deep brain signs were observed in 96% of patients and were usually responsible for death. In general, there was a good correlation between clinical signs and the localization of brain atrophy on MRI. The 'variant' biochemical phenotype characterized by mild abnormalities of the cellular trafficking of endocytosed cholesterol was over-represented in the adult form of NPC and seemed associated with less frequent splenomegaly in childhood and lesser psychiatric signs. Involvement of the NPC1 gene was shown in 33 families and of the NPC2 gene in one. Improving the knowledge of the disease among psychiatrists and neurologists appears essential since emerging treatments should be more efficient at the visceral or cognitive/psychiatric stages of the disease, before the occurrence of widespread deep brain neurological lesions.

Adolescent↗

PubMed2005

Niemann-Pick type C disease: subcellular location and functional characterization of NPC2 proteins with naturally occurring missense mutations.

Niemann-Pick disease type C (NPC), a severe neurovisceral lysosomal disorder, is due to mutations on the NPC1 gene or, in a minority of families, the NPC2 gene. Few investigations have been devoted to the NPC2 protein, for which only 13 different disease-causing mutations (including three novel ones in this report) have been described. Among the currently known NPC2 mutant alleles, six resulted in a premature stop codon. Only five missense mutations, c.115G>A (p.V39M), c.140G>T (p.C47F), c.199T>C (p.S67P), c.278G>T (p.C93F), and (this report) c.295T>C (p.C99R) were identified. In the present study, we generated cDNA constructs harboring each of these missense mutations and, upon overexpression in human fibroblasts with a nonsense NPC2 mutation, characterized the mutated proteins by immunoblotting, immunocytofluorescence microscopy, and complementation. Mutation p.V39M, described in the homozygous state in two patients with an adult-onset neurological disease, resulted in the synthesis of apparently functional recombinant proteins correctly targeted to lysosomes. Although a mild functional impact could possibly be overlooked in our overexpression system, comparative studies with NPC1 mutants indicated that mild mutations might not necessarily affect localization of the protein or its quantity in the native state. Conversely, mutations p.C47F, p.C93R, p.C99R but also, less predictably, p.S67P, led to the synthesis of misfolded recombinant proteins that colocalized with an endoplasmic reticulum marker. The four latter proteins were normally secreted but were unable to correct cholesterol storage in NPC2(-/-) cells. Functional characterization of the mutant proteins showed an excellent genotype-phenotype correlation in the three cases for whom a clinical history was available.

Adult↗

PubMed2004

Structure and function of the NPC2 protein.

Somatic cell hydridization and linkage studies indicated the implication of a second gene as a cause of Niemann-Pick C disease in a minority (5%) of patients. A study of the lysosomal proteome led to the identification of a previously known gene, HE1, as the NPC2 gene. The mature NPC2/HE1 protein is a ubiquitous soluble small 132-amino-acid glycoprotein, first characterized as a major secretory protein in the human epididymis, but also detected in most tissues. Seventeen families with mutations in the NPC2 gene are known. Good genotype-phenotype correlations were observed. No distinction can be made between the biochemical phenotypes of NPC1 or NPC2 mutants. The NPC2 protein binds cholesterol with submicromolar affinity at neutral and acidic pH. The bovine protein has been crystallized, and the cholesterol-binding site assigned to a hydrophobic loosely packed region. There is strong evidence that the NPC1 and NPC2 proteins must function in a closely related fashion. Current data have led to the hypothesis that NPC2 would bind cholesterol from internal lysosomal membranes, enabling a physical interaction with NPC1 (or another protein) and allowing postlysosomal export of cholesterol. In this model, the activity of NPC1 would depend on that of NPC2. The precise function of the NPC2 protein has, however, not been fully elucidated.

Amino Acid Sequence↗

PubMed2004

Niemann-Pick type C disease: importance of N-glycosylation sites for function and cellular location of the NPC2 protein.

Niemann-Pick disease type C (NPC), a neurovisceral disorder characterized by accumulation of cholesterol and glycolipids in the lysosomal/late endosomal system, is due to mutations on either the NPC1 or the NPC2 genes. Although NPC1 and NPC2 proteins appear essential for proper cellular cholesterol trafficking, their precise functions and relationship have remained elusive. Mutation identification in NPC2 patients did not provide insights into structure-function relationships, but recent studies brought important information on the cholesterol-binding site of the NPC2 protein. The present work was focused on localization and N-glycosylation of NPC2, considering that glycosylation is often essential for targeting, stability and biological function of proteins. Using immunocytofluorescence in cultured human fibroblasts, we found that the native NPC2 protein is essentially lysosomal, at variance with the late endosomal location of NPC1. Expression of cDNA mutants affecting each of the three potential NPC2 N-glycosylation sites in NPC2-/- fibroblasts showed that only two sites are used. The intracellular human NPC2 protein occurred as two N-glycosylated forms, with either one single oligosaccharide chain attached to Asn 58 or two oligosaccharides attached to Asn 58 and 135. The oligosaccharidic chains were of the hybrid and/or high mannose type, with no complex chains. Further studies on the cellular location of Asn 58 and Asn 135 mutant proteins and their respective effect on restoration of normal cholesterol traficking in NPC2-/- cells led to the conclusion that only the oligosaccharide chain carried by Asn 58 is responsible for proper targeting of NPC2 to lysosomes, and is crucial for NPC2 function.

Binding Sites↗

PubMedDate not supplied

Niemann-Pick C disease: use of denaturing high performance liquid chromatography for the detection of NPC1 and NPC2 genetic variations and impact on management of patients and families.

Niemann-Pick disease type C (NPC), a neurovisceral disorder characterized by accumulation of unesterified cholesterol and glycolipids in the lysosomal/late endosomal system, is due to mutations on either the NPC1 or the NPC2 genes. While the corresponding proteins appear essential for proper cellular cholesterol trafficking, their precise function and relationship are still unclear. Mutational analysis of patients, useful for the study of structure/function relationships, is especially valuable for proper management of affected families. Correlations have been found between genotypes and the severity of the neurological outcome of the patients, and molecular genetics constitutes the optimal approach for prenatal diagnosis. However, mutation detection in NPC disease is a challenge. The NPC1 gene, affected in >95% of the families, is large in size (approximately 50 kb), and the already known disease-causing mutations and numerous polymorphisms are scattered over 25 exons. Furthermore, detection of NPC2 patients by complex genetic complementation tests is unpractical. In the present study, we describe a rapid and reliable strategy for detecting NPC genetic variations using DHPLC analysis. Conditions of analysis were optimized for all the NPC1 and NPC2 30 exons and validated using 38 previously genotyped patients. These conditions were then applied to screen a panel of 35 genetically uncharacterized, unrelated NPC patients. Pathogenic mutations were identified in 68/70 alleles. Among the mutations identified, 29 were novel, including two of the NPC2 gene. We conclude that DHPLC is a rapid, low-cost, highly accurate, and efficient technique for the detection of NPC genetic variants.

Base Sequence↗