NPC1 Research Articles

Soybean (Glycine max) Haplotype Map (GmHapMap): a universal resource for soybean translational and functional genomics.

SummaryHere, we describe a worldwide haplotype map for soybean (GmHapMap) constructed using whole‐genome sequence data for 1007 Glycine max accessions and yielding 14.9 million variants as well as 4.3 M tag single‐nucleotide polymorphisms (SNPs). When sampling random subsets of these accessions, the number of variants and tag SNPs plateaued beyond approximately 800 and 600 accessions, respectively. This suggests extensive coverage of diversity within the cultivated soybean. GmHapMap variants were imputed onto 21 618 previously genotyped accessions with up to 96% success for common alleles. A local association analysis was performed with the imputed data using markers located in a 1‐Mb region known to contribute to seed oil content and enabled us to identify a candidate causal SNP residing in the NPC1 gene. We determined gene‐centric haplotypes (407 867 GCHs) for the 55 589 genes and showed that such haplotypes can help to identify alleles that differ in the resulting phenotype. Finally, we predicted 18 031 putative loss‐of‐function (LOF) mutations in 10 662 genes and illustrated how such a resource can be used to explore gene function. The GmHapMap provides a unique worldwide resource for applied soybean genomics and breeding.

Evolutionary History of Major Chemosensory Gene Families across Panarthropoda.

Chemosensory perception is a fundamental biological process of particular relevance in basic and applied arthropod research. However, apart from insects, there is little knowledge of specific molecules involved in this system, which is restricted to a few taxa with uneven phylogenetic sampling across lineages. From an evolutionary perspective, onychophorans (velvet worms) and tardigrades (water bears) are of special interest since they represent the closest living relatives of arthropods, altogether comprising the Panarthropoda. To get insights into the evolutionary origin and diversification of the chemosensory gene repertoire in panarthropods, we sequenced the antenna- and head-specific transcriptomes of the velvet worm Euperipatoides rowelli and analyzed members of all major chemosensory families in representative genomes of onychophorans, tardigrades, and arthropods. Our results suggest that the NPC2 gene family was the only family encoding soluble proteins in the panarthropod ancestor and that onychophorans might have lost many arthropod-like chemoreceptors, including the highly conserved IR25a receptor of protostomes. On the other hand, the eutardigrade genomes lack genes encoding the DEG-ENaC and CD36-sensory neuron membrane proteins, the chemosensory members of which have been retained in arthropods; these losses might be related to lineage-specific adaptive strategies of tardigrades to survive extreme environmental conditions. Although the results of this study need to be further substantiated by an increased taxon sampling, our findings shed light on the diversification of chemosensory gene families in Panarthropoda and contribute to a better understanding of the evolution of animal chemical senses.

High diagnostic value of plasma Niemann-Pick type C biomarkers in adults with selected neurological and/or psychiatric disorders.

Late-onset Niemann-Pick type C (NP-C) is a rare, underdiagnosed lysosomal disease with neurological manifestations. A specific treatment, miglustat, can stabilize the disease if given early. Recently, three plasma screening biomarkers (PSBs) were developed [cholestane3β,5α,6βtriol (C-triol), 7-ketocholesterol (7-KC), and lysosphingomyelin-509 (LSM-509)], allowing a simpler and quite robust screening of patients suitable for genetic testing. The objective of our study was to evaluate practical utility and feasibility of large-scale PSB screening for NP-C in selected adult patients. Patients were prospectively enrolled if they showed, starting from 12years of age, at least one of the three initial neuro-psychiatric manifestations described in NP-C: (1) gait disorder (cerebellar and/or dystonic); (2) cognitive decline with frontal lobe syndrome; (3) atypical psychosis. PSBs were measured in plasma of all patients and, if positive (LSM-509 and/or C-triol + 7-KC elevated), sequencing of NPC1 and NPC2 genes was performed. A total of 251 patients [136 males, 115 females; median age 42.1 (range 12.2-85.6) years] were screened. Six patients had positive PSBs. Two were confirmed to have NP-C (0.8% diagnostic yield, both with all three PSBs highly increased, especially LSM-509). False-positive rate was 1.2%, which was identical if only considering LSM-509. By contrast, false-positive rates were 8.1% and 5.7% for 7-KC and C-triol, respectively. We showed that selecting patients with neurologic and/or psychiatric symptoms consistent with NP-C for large-scale PSB screening is a simple and valid strategy to identify new adult NP-C patients, and would probably lead to earlier diagnosis and treatment administration if widely applied.

Identification of Brain-Specific Treatment Effects in NPC1 Disease by Focusing on Cellular and Molecular Changes of Sphingosine-1-Phosphate Metabolism.

Niemann–Pick type C1 (NPC1) is a lysosomal storage disorder, inherited as an autosomal-recessive trait. Mutations in the Npc1 gene result in malfunction of the NPC1 protein, leading to an accumulation of unesterified cholesterol and glycosphingolipids. Beside visceral symptoms like hepatosplenomegaly, severe neurological symptoms such as ataxia occur. Here, we analyzed the sphingosine-1-phosphate (S1P)/S1P receptor (S1PR) axis in different brain regions of Npc1−/− mice and evaluated specific effects of treatment with 2-hydroxypropyl-β-cyclodextrin (HPβCD) together with the iminosugar miglustat. Using high-performance thin-layer chromatography (HPTLC), mass spectrometry, quantitative real-time PCR (qRT-PCR) and western blot analyses, we studied lipid metabolism in an NPC1 mouse model and human skin fibroblasts. Lipid analyses showed disrupted S1P metabolism in Npc1−/− mice in all brain regions, together with distinct changes in S1pr3/S1PR3 and S1pr5/S1PR5 expression. Brains of Npc1−/− mice showed only weak treatment effects. However, side effects of the treatment were observed in Npc1+/+ mice. The S1P/S1PR axis seems to be involved in NPC1 pathology, showing only weak treatment effects in mouse brain. S1pr expression appears to be affected in human fibroblasts, induced pluripotent stem cells (iPSCs)-derived neural progenitor and neuronal differentiated cells. Nevertheless, treatment-induced side effects make examination of further treatment strategies indispensable.

Unbiased yeast screens identify cellular pathways affected in Niemann-Pick disease type C.

Niemann-Pick disease type C (NPC) is a rare lysosomal storage disease caused by mutations in either the NPC1 or NPC2 genes. Mutations in the NPC1 gene lead to the majority of clinical cases (95%); however, the function of NPC1 remains unknown. To gain further insights into the biology of NPC1, we took advantage of the homology between the human NPC1 protein and its yeast orthologue, Niemann-Pick C-related protein 1 (Ncr1). We recreated the NCR1 mutant in yeast and performed screens to identify compensatory or redundant pathways that may be involved in NPC pathology, as well as proteins that were mislocalized in NCR1-deficient yeast. We also identified binding partners of the yeast Ncr1 orthologue. These screens identified several processes and pathways that may contribute to NPC pathogenesis. These included alterations in mitochondrial function, cytoskeleton organization, metal ion homeostasis, lipid trafficking, calcium signalling, and nutrient sensing. The mitochondrial and cytoskeletal abnormalities were validated in patient cells carrying mutations in NPC1, confirming their dysfunction in NPC disease.

Correction of Niemann-Pick type C1 trafficking and activity with the histone deacetylase inhibitor valproic acid

Niemann-Pick type C (NPC) disease is primarily caused by mutations in the NPC1 gene and is characterized by the accumulation of unesterified cholesterol and lipids in the late endosomal (LE) and lysosomal (Ly) compartments. The most prevalent disease-linked mutation is the I1061T variant of NPC1, which exhibits defective folding and trafficking from the endoplasmic reticulum to the LE/Ly compartments. We now show that the FDA-approved histone deacetylase inhibitor (HDACi) valproic acid (VPA) corrects the folding and trafficking defect associated with I1061T-NPC1 leading to restoration of cholesterol homeostasis, an effect that is largely driven by a reduction in HDAC7 expression. The VPA-mediated trafficking correction is in part associated with an increase in the acetylation of lysine residues in the cysteine-rich domain of NPC1. The HDACi-mediated correction is synergistically improved by combining it with the FDA-approved anti-malarial, chloroquine, a known lysosomotropic compound, which improved the stability of the LE/Ly-localized fraction of the I1061T variant. We posit that combining the activity of VPA, to modulate epigenetically the cellular acetylome, with chloroquine, to alter the lysosomal environment to favor stability of the trafficked I1061T variant protein can have a significant therapeutic benefit in patients carrying at least one copy of the I1061T variant of NPC1, the most common disease-associated mutation leading to NPC disease. Given its ability to cross the blood-brain barrier, we posit VPA provides a potential mechanism to improve the response to 2-hydroxypropyl-β-cyclodextrin, by restoring a functional NPC1 to the cholesterol managing compartment as an adjunct therapy.

2-Hydroxypropyl-gamma-cyclodextrin overcomes NPC1 deficiency by enhancing lysosome-ER association and autophagy

Niemann-Pick type C (NPC) disease is a fatal neurodegenerative disorder caused by mutations in NPC1 and NPC2 genes that result in an accumulation of cholesterol in lysosomes. The majority of children with NPC die in adolescence. Currently, no FDA-approved therapies exist for NPC and the mechanisms of NPC disease are not fully understood. Our recent study and the reports from other laboratories showed that 2-hydroxypropyl-γ-cyclodextrin (HPγCD) alleviates cholesterol accumulation in NPC1-deficient cells in spite of its low binding affinity for cholesterol. In this study, we explored the cellular changes that are induced upon HPγCD treatment in NPC1 patient-derived fibroblasts. We show that HPγCD treatment increases lysosome-ER association and enhances autophagic activity. Our study indicates that HPγCD induces an activation of the transcription factor EB (TFEB), a master regulator of lysosomal functions and autophagy. Lysosome-ER association could potentially function as conduits for cholesterol transport from lysosomes to the ER. Accumulating evidence suggests a role for autophagy in rescuing the cholesterol accumulation in NPC and other degenerative diseases. Collectively, our findings suggest that HPγCD restores cellular homeostasis in NPC1-deficient cells via enhancing lysosomal dynamics and functions. Understanding the mechanisms of HPγCD-induced cellular pathways could contribute to effective NPC therapies.

Recent advances in the treatment of Niemann pick disease type C: A mini-review.

Niemann Pick disease Type C (NPC) is a recessive rare disease caused by the mutation on NPC1 and/or NPC2 genes changing the processing of the Low-density proteins (LDL) resulting in an accumulation of lipids in the cells. Until today there is not a cure, the current treatment is based on palliative affairs to reduce the symptoms and prevent its appearance. Among all the treatments proposed the use of cyclodextrins (CDs), nanocarriers which can complex cholesterol, is one of the most useful alternatives. Indeed, for several years 2-hydroxypropyl-β-CD (HPβ-CD) is approved as orphan drug for FDA and EMA to the treatment. However, different CDs based materials are created each year to improve the cholesterol uptake. This review is focused on the novelty of CD based materials for NPC treatment.

Pre-mRNA splicing defects and RNA binding protein involvement in Niemann Pick type C disease

Niemann-Pick type C (NPC) is an autosomal recessive lysosomal storage disorder due to mutations in NPC1 (95 % cases) or NPC2 genes, encoding NPC1 and NPC2 proteins, respectively. Both NPC1 and NPC2 proteins are involved in transport of intracellular cholesterol and their alteration leads to the accumulation of unesterified cholesterol and other lipids within the lysosomes.The disease is characterized by visceral, neurological and psychiatric symptoms. However, the pathogenic mechanisms that lead to the fatal neurodegeneration are still unclear.To date, several mutations leading to the generation of aberrant splicing variants or mRNA degradation in NPC1 and NPC2 genes have been reported. In addition, different lines of experimental evidence have highlighted the possible role of RNA-binding proteins and RNA-metabolism, in the onset and progression of many neurodegenerative disorders, that could explain NPC neurological features and in general, the disease pathogenesis. In this review, we will provide an overview of the impact of mRNA processing and metabolism on NPC disease pathology.

Variants in the NPC1 Gene in Egyptian Patients with Niemann-Pick Type C

BACKGROUND: Niemann-Pick disease type C (NPC) is a rare, autosomal recessive, progressive neuro-visceraldisease caused by biallelic mutations in either NPC1gene (95% of cases) or NPC2 gene.
 AIM: This caseseries study aimed at the molecular analysis of certain hot spots of NPC1 genein NPC Egyptian patients.
 METHODS: The study included 15 unrelated NPC patients and selected parents,as well as20 healthy controls of matched sex and age. Clinical investigations were performed according to well established clinical criteria. Assessment of the chitotriosidase level, as an initial screening tool for NPC, was done in all cases. Polymerase chain reaction amplification of NPC1 exons (17–25) encountering the hotspot residues (855–1098 and1038–1253) was carried out followed by direct sequencingfor mutational analysis.
 RESULTS: All includedpatients with mainly neurovisceral involvement were characterized. The onset of the disease varied from early-infantile (58.3%) to late-infantile (26.7%) and juvenile-onset (6.7%). Ahigh chitotriosidase level wasobservedin all patients. Molecular analysis of NPC1 (exons 17–25) confirmed 15 mutant alleles out of 30 studied ones. They included two novel homozygous missense variants (p.Ser1169Arg and p.Ser1197Phe) and previously reportedfour mutations (p.Arg958*, p.Gly910Ser, p.Ala927Glyfs*38, and andp.Cys1011*).
 CONCLUSION: The two studied amino acid residues (855–1098 and 1038–1253) could beconsidered aspotential hotspot regions in NPC1 Egyptian patients.

Insufficiency of ciliary cholesterol in hereditary Zellweger syndrome

Primary cilia are antenna‐like organelles on the surface of most mammalian cells that receive sonic hedgehog (Shh) signaling in embryogenesis and carcinogenesis. Cellular cholesterol functions as a direct activator of a seven‐transmembrane oncoprotein called Smoothened (Smo) and thereby induces Smo accumulation on the ciliary membrane where it transduces the Shh signal. However, how cholesterol is supplied to the ciliary membrane remains unclear. Here, we report that peroxisomes are essential for the transport of cholesterol into the ciliary membrane. Zellweger syndrome (ZS) is a peroxisome‐deficient hereditary disorder with several ciliopathy‐related features and cells from these patients showed a reduced cholesterol level in the ciliary membrane. Reverse genetics approaches revealed that the GTP exchange factor Rabin8, the Rab GTPase Rab10, and the microtubule minus‐end‐directed kinesin KIFC3 form a peroxisome‐associated complex to control the movement of peroxisomes along microtubules, enabling communication between peroxisomes and ciliary pocket membranes. Our findings suggest that insufficient ciliary cholesterol levels may underlie ciliopathies.

Ultrastructure of spinal anterior horn cells in human Niemann‐Pick type C (NPC) patient and mouse model of NPC with retroposon insertion in NPC1 genes

Niemann-Pick disease type C (NPC) is a neurovisceral lipid-storage disease. Although NPC patients show lipid storage in anterior horn cells of the spinal cord, little information is available regarding the electron microscopic analyses of the morphologies of intra-endosomal lipid like-materials in the anterior horn cells of NPC patients. In this study, we elucidated the intra-endosomal ultrastructures in spinal anterior horn cells in an NPC patient, as well as in mutant BALB/c NPC1-/- mice with a retroposon insertion in the NPC1 gene. These morphologies were classified into four types: vesicle, multiple concentric sphere (MCS), membrane, and rose flower. The percentages of the composition in the NPC patient and NPC1-/- mice were: vesicle (55.5% and 14.9%), MCS (15.7% and 3.4%), membrane (23.6% and 57.1%), and rose flower (5.2% and 24.6%), respectively. Formation of the intra-endosomal structures could proceed as follows: (i) a vesicle or MCS buds off the endosome into the lumen; (ii) when a vesicle breaks down, a membrane is formed; and (iii) after an MCS breaks down, a rose flower structure is formed. Our new finding in this study is that ultrastructural morphology is the same between the NPC patient and NPC1-/- mice, although there are differences in the composition.

Novel compound heterozygous mutation in NPC1 gene cause Niemann–Pick disease type C with juvenile onset

Niemann-Pick disease type C (NPC) is a progressive lysosomal storage disorder caused by mutations in the NPC1 (in 95% of cases) or NPC2 (in ~5% of cases) genes, inherited in an autosomal recessive manner. We report the case of a 38-year-old woman with learning disorder from her first year of schooling, and could notice slow progressed cognitive impairment, social withdrawal, apathy, handwriting alterations, deterioration of language skills and dysphagia. Brain magnetic resonance imaging showed severe cerebellar atrophy, hypoplasia of the corpus callosum, asymmetric lateral ventricular enlargement, and severe enlargement of frontal and parietal subarachnoid spaces. Next generation sequencing for NPC genes (NPC1 and NPC2) detected compound heterozygous mutations in NPC1 gene, including c.1553G[A (p.Arg518Gln), paternally inherited, and c.1270C[T (p.Pro424Ser) maternally inherited. The first mutation has been already described in literature and correlated to NPC, while the second mutation is still unknown. Moreover, filipin test and quantification of plasma oxysterols confirmed NPC diagnosis. We can suggest the missense mutation c.1270C[T (p.Pro424Ser) as a new causative mutation of NPC.

Transcript, protein, metabolite and cellular studies in skin fibroblasts demonstrate variable pathogenic impacts of NPC1 mutations

BackgroundNiemann-Pick type C (NP-C) is a rare neurovisceral genetic disorder caused by mutations in the NPC1 or the NPC2 gene. NPC1 is a multipass-transmembrane protein essential for egress of cholesterol from late endosomes/lysosomes. To evaluate impacts of NPC1 mutations, we examined fibroblast cultures from 26 NP-C1 patients with clinical phenotypes ranging from infantile to adult neurologic onset forms. The cells were tested with multiple assays including NPC1 mRNA expression levels and allele expression ratios, assessment of NPC1 promoter haplotypes, NPC1 protein levels, cellular cholesterol staining, localization of the mutant NPC1 proteins to lysosomes, and cholesterol/cholesteryl ester ratios. These results were correlated with phenotypes of the individual patients.ResultsOverall we identified 5 variant promoter haplotypes. Three of them showed reporter activity decreased down to 70% of the control sequence. None of the haplotypes were consistently associated with more severe clinical presentation of NP-C. Levels of transcripts carrying null NPC1 alleles were profoundly lower than levels of the missense variants. Low levels of the mutant NPC1 protein were identified in most samples. The protein localised to lysosomes in cultures expressing medium to normal NPC1 levels. Fibroblasts from patients with severe infantile phenotypes had higher cholesterol levels and higher cholesterol/cholesteryl ester ratios. On the contrary, cell lines from patients with juvenile and adolescent/adult phenotypes showed values comparable to controls.ConclusionNo single assay fully correlated with the disease severity. However, low residual levels of NPC1 protein and high cholesterol/cholesteryl ester ratios associated with severe disease. The results suggest not only low NPC1 expression due to non-sense mediated decay or low mutant protein stability, but also dysfunction of the stable mutant NPC1 as contributors to the intracellular lipid transport defect.

Complement Component C3 Participates in Early Stages of Niemann-Pick C Mouse Liver Damage.

Niemann–Pick type C (NPC), a lysosomal storage disorder, is mainly caused by mutations in the NPC1 gene. Niemann–Pick type C patients and mice show intracellular cholesterol accumulation leading to hepatic failure with increased inflammatory response. The complement cascade, which belongs to the innate immunity response, recognizes danger signals from injured tissues. We aimed to determine whether there is activation of the complement system in the liver of the NPC mouse and to assess the relationship between C3 activation, a final component of the pathway, and NPC liver pathology. Niemann–Pick type C mice showed high levels of C3 staining in the liver which unexpectedly decreased with aging. Using an inducible NPC1 hepatocyte rescue mouse model, we restored NPC1 expression for a short time in young mice. We found C3 positive cells only in non-rescued cells, suggesting that C3 activation in NPC cells is reversible. Then, we studied the effect of C3 ablation on NPC liver damage at two postnatal time points, P56 and P72. Deletion of C3 reduced the presence of hepatic CD68-positive cells at postnatal day 56 and prevented the increase of transaminase levels in the blood of NPC mice. These positive effects were abrogated at P72, indicating that the complement cascade participates only during the early stages of liver damage in NPC mice, and that its inhibition may serve as a new potential therapeutic strategy for the disease.

Different Trafficking Phenotypes of Niemann-Pick C1 Gene Mutations Correlate with Various Alterations in Lipid Storage, Membrane Composition and Miglustat Amenability.

Niemann-Pick Type C (NPC) is an autosomal recessive lysosomal storage disease leading to progressive neurodegeneration. Mutations in the NPC1 gene, which accounts for 95% of the cases, lead to a defect in intra-lysosomal trafficking of cholesterol and an accumulation of storage material including cholesterol and sphingolipids in the endo-lysosomal system. Symptoms are progressive neurological and visceral deterioration, with variable onset and severity of the disease. This study investigates the influence of two different NPC1 mutations on the biochemical phenotype in fibroblasts isolated from NPC patients in comparison to healthy, wild type (WT) cells. Skin derived fibroblasts were cultured from one patient compound-heterozygous for D874V/D948Y mutations, which presented wild-type like intracellular trafficking of NPC1, and a second patient compound- heterozygous for I1061T/P887L mutations, which exhibited a more severe biochemical phenotype as revealed in the delayed trafficking of NPC1. Biochemical analysis using HPLC and TLC indicated that lipid accumulations were mutation-dependent and correlated with the trafficking pattern of NPC1: higher levels of cholesterol and glycolipids were associated with the mutations that exhibited delayed intracellular trafficking, as compared to their WT-like trafficked or wild type (WT) counterparts. Furthermore, variations in membrane structure was confirmed in these cell lines based on alteration in lipid rafts composition as revealed by the shift in flotillin-2 (FLOT2) distribution, a typical lipid rafts marker, which again showed marked alterations only in the NPC1 mutant showing major trafficking delay. Finally, treatment with N-butyldeoxynojirimycin (NB-DNJ, Miglustat) led to a reduction of stored lipids in cells from both patients to various extents, however, no normalisation in lipid raft structure was achieved. The data presented in this study help in understanding the varying biochemical phenotypes observed in patients harbouring different mutations, which explain why the effectiveness of NB-DNJ treatment is patient specific.

Gene therapy to the blood-brain barrier with resulting protein secretion as a strategy for treatment of Niemann Picks type C2 disease.

Treatment of many diseases affecting the central nervous system (CNS) is complicated by the inability of several therapeutics to cross the blood-brain barrier (BBB). Genetically modifying brain capillary endothelial cells (BCECs) denotes an approach to overcome the limitations of the BBB by turning BCECs into recombinant protein factories. This will result in protein secretion toward both the brain and peripheral circulation, which is particularly relevant in genetic diseases, like lysosomal storage diseases (LSD), where cells are ubiquitously affected both in the CNS and the periphery. Here we investigated transfection of primary rat brain capillary endothelial cells (rBCECs) for synthesis and secretion of recombinant NPC2, the protein deficient in the lysosomal cholesterol storage disease NiemannPick type C2. We demonstrate prominent NPC2 gene induction and protein secretion in 21% of BCECs in non-mitotic monocultures with a biological effect on NPC2-deficient fibroblasts as verified from changes in filipin III staining of cholesterol deposits. By comparison the transfection efficiency was 75% in HeLa-cells, known to persist in a mitotic state. When co-cultured with primary rat astrocytes in conditions with maintained BBB properties 7% BCECs were transfected, clearly suggesting that induction of BBB properties with polarized conditions of the non-mitotic BCECs influences the transfection efficacy and secretion directionality. In conclusion, non-viral gene therapy to rBCECs leads to protein secretion and signifies a method for NPC2 to target cells inside the CNS otherwise inaccessible because of the presence of the BBB. However, obtaining high transfection efficiencies is crucial in order to achieve sufficient therapeutic effects. Cover Image for this issue: https://doi.org/10.1111/jnc.15050.

Molecular Genetics of Niemann-Pick Type C Disease in Italy: An Update on 105 Patients and Description of 18 NPC1 Novel Variants.

Niemann-Pick type C (NPC) disease is an autosomal recessive lysosomal storage disorder caused by mutations in NPC1 or NPC2 genes. In 2009, the molecular characterization of 44 NPC Italian patients has been published. Here, we present an update of the genetic findings in 105 Italian NPC patients belonging to 83 unrelated families (77 NPC1 and 6 NPC2). NPC1 and NPC2 genes were studied following an algorithm recently published. Eighty-four different NPC1 and five NPC2 alleles were identified. Only two NPC1 alleles remained non detected. Sixty-two percent of NPC1 alleles were due to missense variants. The most frequent NPC1 mutation was the p.F284Lfs*26 (5.8% of the alleles). All NPC2 mutations were found in the homozygous state, and all but one was severe. Among newly diagnosed patients, 18 novel NPC1 mutations were identified. The pathogenic nature of 7/9 missense alleles and 3/4 intronic variants was confirmed by filipin staining and NPC1 protein analysis or mRNA expression in patient’s fibroblasts. Taken together, our previous published data and new results provide an overall picture of the molecular characteristics of NPC patients diagnosed so far in Italy.

Niemann-Pick type C1 regulates cholesterol transport and metamorphosis in silkworm, Bombyx mori (Dazao)

NPC1 is a large glycoprotein with 13 transmembrane-spanning domains, which plays a crucial biological role in cholesterol transport and metamorphosis of animals. However, the physiological functions of this gene have rarely been elucidated in insects. Here, we isolated the NPC1 gene from Bombyx mori (BmNPC1), sequenced and evaluated its physiological functions. BmNPC1 comprised of 3702 bp open reading frame, encoding a protein of 1233 amino acid residues. The recombinant protein was expressed, and anti-BmNPC1 antibodies were synthesized. Immunofluorescence assay revealed that BmNPC1 protein localized in the cytoplasm of the cells. The qRT-PCR analysis showed that BmNPC1 expression was most significant in the testis, followed by the malpighian tubules, hemocytes, and ovary. The knockdown of BmNPC1 by double-stranded RNA caused the accumulation of cholesterol in the cells. Furthermore, suppression of this gene influenced the expression of ecdysone-responsive genes and also prevented the molting in B. mori (Dazao) larvae. Overall, BmNPC1 may have different biological roles in the physiology of silkworm, B. mori (Dazao), since it regulates the cholesterol transport and molting process.

New variants in Spanish Niemann-Pick type c disease patients.

Niemann-Pick type C (NPC) disease is a rare inherited disease, with progressive neurodegeneration as the main symptom. It is a lysosomal storage disorder caused by mutations in NPC1 or NPC2 genes, leading to a lysosomal cholesterol trafficking impairment. Disease indicators are the clinical suspicion and biomarker levels. However, a genetic study is mandatory for the diagnosis, which is complicated due to the different variants with unknown significance. The aim of this work was to identify the variants responsible for NPC in our pediatric population. Twenty-two samples from non-related infants believed to have NPC disease were analyzed during the last 3years. Surrogate biomarkers of the disease were evaluated whenever possible. Sanger sequencing for both genes is reported for all samples. Complementary genetic studies were performed when necessary. NPC disease was confirmed in 31.8% of subjects due to homozygous or compound heterozygous genetic variants in NPC1. The following four novel variants were identified: a gross deletion variant composed of the gene promoter and the first exon, NM_000271.3:c.385delT, NM_000271.3:c.1553+1342_1655-291del, and NM_000271.3:c.1757delA. None had functional activity and all resulted in important structural changes in the protein.