Laboratory diagnosis of Niemann–Pick disease type C: The filipin staining test
TL;DR: Methodological caveats and variability of patterns encountered in patients with proven Niemann-Pick C disease (typical "classic" or "intermediate," atypical "variant") are described, leading to a proposed algorithm for interpretation of results in the filipin test.
Abstract: Niemann-Pick disease type C (NPC) is an atypical neurovisceral lysosomal storage disorder resulting from mutations in either the NPC1 or the NPC2 gene, currently conceived as a lipid trafficking disorder. Impaired egress of cholesterol from the late endosomal/lysosomal (LE/L) compartment is a key element of the pathogenesis. The resulting accumulation of unesterified cholesterol in the LE/L compartment can be visualized by fluorescence microscopy after staining with filipin. The "filipin test," performed on cultured fibroblasts, is the historical gold standard method to establish the diagnosis in patients. The authors provide methodological details of the protocol developed and used in their laboratory since 1988, in which two sources of low-density lipoproteins (LDL) (total serum and pure LDL) are used in parallel to facilitate the final interpretation. Methodological caveats and variability of patterns encountered in patients with proven Niemann-Pick C disease (typical "classic" or "intermediate," atypical "variant") are described. An overview of the past 5 years referrals (533 subjects tested, 57 NPC cases, but also 74 mildly/weakly positive tests not due to NPC) is discussed, leading to a proposed algorithm for interpretation of results in the filipin test. This tool takes into account the limits of the method. In up to 15% of all referrals, the filipin test was inconclusive in absence of molecular analysis. Patients diagnosed in the adult age preferentially showed an "intermediate" or "variant" pattern. Well conducted, the filipin test remains an efficient approach for diagnosing NPC, and it is a good functional test to study the pathogenicity of novel mutations.
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TL;DR: A reappraisal of lipid storage and lysosomal enzymes activities in tissues/cells from NPC patients and animal models is provided, with emphasis on differences between systemic organs and the brain.
Abstract: Niemann-Pick disease type C (NPC) is an atypical lysosomal storage disease resulting from mutations in one of two genes, either NPC1 or NPC2. Although a neurovisceral disorder, it is above all a neurodegenerative disease in the vast majority of patients. Not an enzyme deficiency, it is currently conceived as a lipid trafficking disorder. Impaired egress of cholesterol from the late endosomal/lysosomal (LE/L) compartment is a specific and key element of the pathogenesis, but other lipids, more specially sphingolipids, are also involved, and there are indications for further abnormalities. The full function of the NPC1 and NPC2 proteins is still unclear. This review provides a reappraisal of lipid storage and lysosomal enzymes activities in tissues/cells from NPC patients and animal models. It summarizes the current knowledge on the NPC1 and NPC2 proteins and their function in transport of cholesterol within the late endosomal-lysosomal compartment, with emphasis on differences between systemic organs and the brain; it also discusses regulation by membrane lipids of the NPC2-mediated cholesterol trafficking, interplay between cholesterol and sphingomyelin, the metabolic origin of glycosphingolipids stored in brain, and the putative role of free sphingoid bases in pathogenesis. Brief mention is finally made of diseases affecting other genes that were very recently shown to impact the "NPC pathway".
213 citations
Cites background from "Laboratory diagnosis of Niemann–Pic..."
...…observation that the "variant" phenotype was more frequent in the adult or juvenile neurological onset forms, rare in the late infantile form and not seen (possibly exceptional) in the early infantile form (Vanier et al 1991b; Vanier and Suzuki 1998) still holds today (Vanier and Latour 2015)....
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University of Birmingham1, Royal Free London NHS Foundation Trust2, Vancouver General Hospital3, Hospital Sant Joan de Déu Barcelona4, University of Lyon5, Royal Melbourne Hospital6, University Hospitals Birmingham NHS Foundation Trust7, University of Paris8, Salford Royal NHS Foundation Trust9, University of Barcelona10, University Hospital Regensburg11, Central Manchester University Hospitals NHS Foundation Trust12, University of Washington13, Mayo Clinic14
TL;DR: Clinical guidelines that define standard of care for NPC patients, foster shared care arrangements between expert centres and family physicians, and empower patients are developed that can inform care providers, care funders, patients and their carers of best practice of care.
Abstract: Niemann-Pick Type C (NPC) is a progressive and life limiting autosomal recessive disorder caused by mutations in either the NPC1 or NPC2 gene. Mutations in these genes are associated with abnormal endosomal-lysosomal trafficking, resulting in the accumulation of multiple tissue specific lipids in the lysosomes. The clinical spectrum of NPC disease ranges from a neonatal rapidly progressive fatal disorder to an adult-onset chronic neurodegenerative disease. The age of onset of the first (beyond 3 months of life) neurological symptom may predict the severity of the disease and determines life expectancy.NPC has an estimated incidence of ~ 1: 100,000 and the rarity of the disease translate into misdiagnosis, delayed diagnosis and barriers to good care. For these reasons, we have developed clinical guidelines that define standard of care for NPC patients, foster shared care arrangements between expert centres and family physicians, and empower patients. The information contained in these guidelines was obtained through a systematic review of the literature and the experiences of the authors in their care of patients with NPC. We adopted the Appraisal of Guidelines for Research & Evaluation (AGREE II) system as method of choice for the guideline development process. We made a series of conclusive statements and scored them according to level of evidence, strengths of recommendations and expert opinions. These guidelines can inform care providers, care funders, patients and their carers of best practice of care for patients with NPC. In addition, these guidelines have identified gaps in the knowledge that must be filled by future research. It is anticipated that the implementation of these guidelines will lead to a step change in the quality of care for patients with NPC irrespective of their geographical location.
194 citations
Cites background from "Laboratory diagnosis of Niemann–Pic..."
...Until recently, the demonstration of unesterified cholesterol accumulation within the lysosomes by filipin testing was considered to be the gold standard test for the diagnosis of NPC disease [11, 47, 51]....
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...Additionally, the assay is technically challenging, labour intensive and is performed only in specialized laboratories [51]....
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TL;DR: Advances in mass spectrometry has allowed identification of several sensitive plasma biomarkers elevated in NP-C, which, together with the concomitant progress in molecular genetic technology, have greatly impacted the strategy of laboratory testing.
115 citations
Cites background from "Laboratory diagnosis of Niemann–Pic..."
...Results from ‘variant’ or inconclusive tests must be evaluated in light of the clinical presentation, and complemented by sequencing of the NPC1 and NPC2 genes [10,23]....
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...Genetic analysis is necessary to confirm a diagnosis of NP-C following abnormal results with disease biomarkers or inconclusive filipin staining results [10,23], but identification of NPC1 or NPC2 mutations should also optimally complement even clear-cut results from filipin staining [10]....
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...Other limitations include the requirement for costly LPDS and LDL reagents, the need to include positive and negative control cells in all experiments and, considering the pitfalls outlined above, the necessary experience in interpretation to ensure accurate results [23]....
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...In case of a ‘variant’ pattern, complementary sequencing of the NPC1 and NPC2 genes is recommended before making a conclusive diagnosis [10,23]....
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...This pattern has been described as the ‘variant’ profile [20, 23]....
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TL;DR: It is shown that tamoxifen resistance cannot simply be explained by altered expression of individual genes, common mechanism across all resistant variants, or the appearance of new fusion genes, and novel drug vulnerabilities associated with this phenotype are suggested.
Abstract: Tamoxifen treatment of estrogen receptor (ER)-positive breast cancer reduces mortality by 31%. However, over half of advanced ER-positive breast cancers are intrinsically resistant to tamoxifen and about 40% will acquire the resistance during the treatment. In order to explore mechanisms underlying endocrine therapy resistance in breast cancer and to identify new therapeutic opportunities, we created tamoxifen-resistant breast cancer cell lines that represent the luminal A or the luminal B. Gene expression patterns revealed by RNA-sequencing in seven tamoxifen-resistant variants were compared with their isogenic parental cells. We further examined those transcriptomic alterations in a publicly available patient cohort. We show that tamoxifen resistance cannot simply be explained by altered expression of individual genes, common mechanism across all resistant variants, or the appearance of new fusion genes. Instead, the resistant cell lines shared altered gene expression patterns associated with cell cycle, protein modification and metabolism, especially with the cholesterol pathway. In the tamoxifen-resistant T-47D cell variants we observed a striking increase of neutral lipids in lipid droplets as well as an accumulation of free cholesterol in the lysosomes. Tamoxifen-resistant cells were also less prone to lysosomal membrane permeabilization (LMP) and not vulnerable to compounds targeting the lipid metabolism. However, the cells were sensitive to disulfiram, LCS-1, and dasatinib. Altogether, our findings highlight a major role of LMP prevention in tamoxifen resistance, and suggest novel drug vulnerabilities associated with this phenotype.
96 citations
TL;DR: It is shown that compounds interfering with intracellular cholesterol transport have the capacity to inhibit CHIKV replication in human skin fibroblasts, a major viral entry site in the human host.
Abstract: Chikungunya virus (CHIKV) is an emerging arbovirus of the Togaviridae family that poses a present worldwide threat to human in the absence of any licensed vaccine or antiviral treatment to control viral infection. Here, we show that compounds interfering with intracellular cholesterol transport have the capacity to inhibit CHIKV replication in human skin fibroblasts, a major viral entry site in the human host. Pretreatment of these cells with the class II cationic amphiphilic compound U18666A, or treatment with the FDA-approved antidepressant drug imipramine resulted in a near total inhibition of viral replication and production at the highest concentration used without any cytotoxic effects. Imipramine was found to affect both the fusion and replication steps of the viral life cycle. The key contribution of cholesterol availability to the CHIKV life cycle was validated further by the use of fibroblasts from Niemann-Pick type C (NPC) patients in which the virus was unable to replicate. Interestingly, imipramine also strongly inhibited the replication of several Flaviviridae family members, including Zika, West Nile and Dengue virus. Together, these data show that this compound is a potential drug candidate for anti-arboviral treatment.
75 citations
References
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01 Jan 1995
TL;DR: In this paper, the authors present a list of disorders of MITOCHONDRIAL FUNCTION, including the following: DISORDERS OF MIOCHONDRIC FERTILITY XIX, XVI, XIX.
Abstract: I. INTRODUCTION II. PERSPECTIVES III. GENERAL THEMES IV. CANCER V. CHROMOSOMES VI. DIAGNOSTIC APPROACHES VII. CARBOHYDRATES VIII. AMINO ACIDS IX. ORGANIC ACIDS X. DISORDERS OF MITOCHONDRIAL FUNCTION XI. PURINES AND PYRIMIDINES XII. LIPIDS XIII. PORPHYRINS XIV. METALS XV. PEROXISOMES XVI. LYSOSOMAL DISORDERS XVII. VITAMINS XVIII. HORMONES XIX. BLOOD XX. IMMUNE AND DEFENSE SYSTEMS XXI. MEMBRANE TRANSPORT DISORDERS XXII. CONNECTIVE TISSUE XXIII. CARDIOVASCULAR SYSTEM XXIV. KIDNEY XXV. MUSCLE XXVI. LUNG XXVII. SKIN XXVIII. NEUROGENETICS XXIX. EYE XXX. MULTISYSTEM INBORN ERRORS OF DEVELOPMENT
10,525 citations
TL;DR: The relatively low density of the lipoproteins was utilized by Lindgren, Elliott, and Gofman to separate them from the other serum proteins by ultracentrifugal flotation, and quantitation was subsequently performed by refractometric methods in the analytical ultracentRifuge.
Abstract: In the past few years several methods have been developed for the analysis of serum lipoproteins Lindgren, Elliott, and Gofman (1) have utilized the relatively low density of the lipoproteins to separate them from the other serum proteins by ultracentrifugal flotation Quantitation was subsequently performed by refractometric methods in the analytical ultracentrifuge Separations of lipoproteins have also been made by Cohn fractionation in cold ethanol, and the quantities of lipoprotein have been estimated from the lipid content of the fractions (2, 3) Widely used at the present time is the method of zone electrophoresis with quantitation either by staining (4) or by chemical analysis of eluates from the support
8,544 citations
"Laboratory diagnosis of Niemann–Pic..." refers background in this paper
...The theoretical equations allowing calculations of the volume of stock solution to add to adjust the density in the sequential steps of preparation are described in detail in (Havel et al., 1955); they are simplified below....
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...The theoretical equations allowing calculations of the volume of stock solution to add to adjust the density in the sequential steps of preparation are described in detail in (Havel et al., 1955); they are simplified below....
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TL;DR: Study of the cell surface binding, internalization, and metabolism of low-density lipoprotein (LDL) in cultured cells have provided useful information regarding the general aspects of receptor-mediated endocytosis and three classes of mutant alleles at the LDL receptor locus have been deduced.
Abstract: Publisher Summary Studies of the cell surface binding, internalization, and metabolism of low-density lipoprotein (LDL) in cultured cells have provided useful information regarding the general aspects of receptor-mediated endocytosis. The study of the LDL receptor has been facilitated by analysis of mutant fibroblasts obtained from human subjects with disorders of cholesterol metabolism. The most informative cells, obtained from patients with familial hypercholesterolemia (FH), have defects in the gene encoding the LDL receptor. The existence of three classes of mutant alleles at the LDL receptor locus has been deduced on the basis of genetic and kinetic data. One of these alleles specifies a receptor that is unable to bind LDL. The second type of allele specifies a receptor that can bind small amounts of LDL; and the third type of allele specifies a receptor that can bind LDL but cannot be incorporated into coated pits and hence cannot carry the LDL into the cell. The first two alleles are common among FH patients, whereas the third allele is extremely rare.
1,589 citations
"Laboratory diagnosis of Niemann–Pic..." refers background or methods in this paper
...Ultracentrifuge at approximately 200,000 g for 48 h atþ4 C (Goldstein et al., 1983) (e.g., 48 h at 45,000 rpm using a 50.2 Ti Beckman rotor)....
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...Ultracentrifuge at approximately 200,000 g for 48 h atþ4 C (Goldstein et al., 1983) (e....
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TL;DR: The primary laboratory diagnosis requires living skin fibroblasts to demonstrate accumulation of unesterified cholesterol in perinuclear vesicles (lysosomes) after staining with filipin, and genotyping of patients is useful to confirm the diagnosis in the latter patients and essential for future prenatal diagnosis.
Abstract: Niemann-Pick C disease (NP-C) is a neurovisceral atypical lysosomal lipid storage disorder with an estimated minimal incidence of 1/120 000 live births. The broad clinical spectrum ranges from a neonatal rapidly fatal disorder to an adult-onset chronic neurodegenerative disease. The neurological involvement defines the disease severity in most patients but is typically preceded by systemic signs (cholestatic jaundice in the neonatal period or isolated spleno- or hepatosplenomegaly in infancy or childhood). The first neurological symptoms vary with age of onset: delay in developmental motor milestones (early infantile period), gait problems, falls, clumsiness, cataplexy, school problems (late infantile and juvenile period), and ataxia not unfrequently following initial psychiatric disturbances (adult form). The most characteristic sign is vertical supranuclear gaze palsy. The neurological disorder consists mainly of cerebellar ataxia, dysarthria, dysphagia, and progressive dementia. Cataplexy, seizures and dystonia are other common features. NP-C is transmitted in an autosomal recessive manner and is caused by mutations of either the NPC1 (95% of families) or the NPC2 genes. The exact functions of the NPC1 and NPC2 proteins are still unclear. NP-C is currently described as a cellular cholesterol trafficking defect but in the brain, the prominently stored lipids are gangliosides. Clinical examination should include comprehensive neurological and ophthalmological evaluations. The primary laboratory diagnosis requires living skin fibroblasts to demonstrate accumulation of unesterified cholesterol in perinuclear vesicles (lysosomes) after staining with filipin. Pronounced abnormalities are observed in about 80% of the cases, mild to moderate alterations in the remainder ("variant" biochemical phenotype). Genotyping of patients is useful to confirm the diagnosis in the latter patients and essential for future prenatal diagnosis. The differential diagnosis may include other lipidoses; idiopathic neonatal hepatitis and other causes of cholestatic icterus should be considered in neonates, and conditions with cerebellar ataxia, dystonia, cataplexy and supranuclear gaze palsy in older children and adults. Symptomatic management of patients is crucial. A first product, miglustat, has been granted marketing authorization in Europe and several other countries for specific treatment of the neurological manifestations. The prognosis largely correlates with the age at onset of the neurological manifestations.
974 citations
TL;DR: Treatment of NP-C2 fibroblasts with exogenous recombinant HE1 protein ameliorated lysosomal accumulation of low density lipoprotein-derived cholesterol.
Abstract: Niemann-Pick type C2 disease (NP-C2) is a fatal hereditary disorder of unknown etiology characterized by defective egress of cholesterol from lysosomes. Here we show that the disease is caused by a deficiency in HE1, a ubiquitously expressed lysosomal protein identified previously as a cholesterol-binding protein. HE1 was undetectable in fibroblasts from NP-C2 patients but present in fibroblasts from unaffected controls and NP-C1 patients. Mutations in the HE1 gene, which maps to chromosome 14q24.3, were found in NP-C2 patients but not in controls. Treatment of NP-C2 fibroblasts with exogenous recombinant HE1 protein ameliorated lysosomal accumulation of low density lipoprotein-derived cholesterol.
817 citations
"Laboratory diagnosis of Niemann–Pic..." refers background in this paper
...The NPC2 protein is targeted to the lysosome by the mannose-6-phosphate receptor pathway and is nonfunctional if this routing is defective (Naureckiene et al., 2000; Chikh, Vey, Simonot, Vanier, & Millat, 2004; Willenborg et al., 2005), which likely explains this finding....
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