Cancer-associated fibroblasts (CAFs) can play an important role in tumor growth by creating a tumor-promoting microenvironment. Models to study the role of CAFs in the tumor microenvironment can be helpful for understanding the functional importance of fibroblasts, fibroblasts from different tissues, and specific genetic factors in fibroblasts. Mouse models are essential for understanding the contributors to tumor growth and progression in an in vivo context. Here, a protocol in which cancer cells are mixed with fibroblasts and introduced into mice to develop tumors is provided. Tumor sizes over time and final tumor weights are determined and compared among groups. The protocol described can provide more insight into the functional role of CAFs in tumor growth and progression.
NADPH is a critical metabolite that is important for regenerating reduced glutathione from oxidized glutathione and eliminating reactive oxygen species (ROS). Metabolic flux and microarray experiments in fibroblasts demonstrated that NADPH‐producing enzymes, glucose‐6‐phosphate dehydrogenase (G6PD), isocitrate dehydrogenase (IDH), and the upstream transcription factor NRF2, are all activated in quiescent compared with proliferating fibroblasts. We demonstrated that G6PD and NRF2 are functionally important for quiescence by showing that inhibition of G6PD or NRF2 results in oxidative stress and apoptosis specifically in quiescent fibroblasts. Flow cytometry experiments demonstrated that ROS in quiescent cells can be derived from mitochondrial superoxide that results from increased mitochondrial activity in the serum‐starved compared with proliferating fibroblasts, as well as an increase in reactive nitrogen species that arise from peroxisomes. To understand the physiological role of NADPH‐production pathways, we examined their potential activity in mouse skin. Consistent with our findings in quiescent fibroblasts, in situ metabolic activity assays revealed higher potential activity for G6PD and IDH in non‐dividing cells. Of particular interest was the high IDH potential in quiescent hair follicle stem cells. Staining of live mouse skin with monochlorobimane showed higher reactivity, consistent with higher levels of reduced glutathione, in hair follicle stem cells. Inhibition of IDH activity in healthy mouse skin with two different inhibitors resulted in progression in the hair follicle cycle from a quiescent to proliferative state suggesting that the high IDH activity observed in hair follicle stem cells may contribute to the maintenance of these stem cells in a quiescent state. Going forward, our focus is on understanding the role of IDH in the maintenance of stem cells as opposed to proliferating, committed progenitor cells. Support or Funding Information H.A.C. was the Milton E. Cassel scholar of the Rita Allen Foundation. This work was supported by NIGMS Center of Excellence grant P50 GM071508, two grants from the Cancer Institute of New Jersey, the New Jersey Commission on Cancer Research, National Cancer Institute 1RC1 CA147961‐01, a Focused Funding Grant to H.A.C. from the Johnson & Johnson Foundation and a grant from the PhRMA Foundation to H.A.C, and XXXX to W.E.L. J.M.S. was supported by NIH training grant T32 HG003284. E.M.H. acknowledges a Bowen Fellowship from Princeton University and the New Jersey Commission on Cancer Research. E.J.S. acknowledges support from the National Science Foundation.
Niemann-Pick type C1 disease (NPC1) is an autosomal recessive lysosomal storage disorder characterized by neonatal jaundice, hepatosplenomegaly, and progressive neurodegeneration. The present study provides the lipid profiles, mutations, and corresponding associations with the biochemical phenotype obtained from NPC1 patients who participated in the National NPC1 Disease Database. Lipid profiles were obtained from 34 patients (39%) in the survey and demonstrated significantly reduced plasma LDL cholesterol (LDL-C) and increased plasma triglycerides in the majority of patients. Reduced plasma HDL cholesterol (HDL-C) was the most consistent lipoprotein abnormality found in male and female NPC1 patients across age groups and occurred independent of changes in plasma triglycerides. A subset of 19 patients for whom the biochemical severity of known NPC1 mutations could be correlated with their lipid profile showed a strong inverse correlation between plasma HDL-C and severity of the biochemical phenotype. Gene mutations were available for 52 patients (59%) in the survey, including 52 different mutations and five novel mutations (Y628C, P887L, I923V, A1151T, and 3741_3744delACTC). Together, these findings provide novel information regarding the plasma lipoprotein changes and mutations in NPC1 disease, and suggest plasma HDL-C represents a potential biomarker of NPC1 disease severity. Niemann-Pick type C1 disease (NPC1) is an autosomal recessive lysosomal storage disorder characterized by neonatal jaundice, hepatosplenomegaly, and progressive neurodegeneration. The present study provides the lipid profiles, mutations, and corresponding associations with the biochemical phenotype obtained from NPC1 patients who participated in the National NPC1 Disease Database. Lipid profiles were obtained from 34 patients (39%) in the survey and demonstrated significantly reduced plasma LDL cholesterol (LDL-C) and increased plasma triglycerides in the majority of patients. Reduced plasma HDL cholesterol (HDL-C) was the most consistent lipoprotein abnormality found in male and female NPC1 patients across age groups and occurred independent of changes in plasma triglycerides. A subset of 19 patients for whom the biochemical severity of known NPC1 mutations could be correlated with their lipid profile showed a strong inverse correlation between plasma HDL-C and severity of the biochemical phenotype. Gene mutations were available for 52 patients (59%) in the survey, including 52 different mutations and five novel mutations (Y628C, P887L, I923V, A1151T, and 3741_3744delACTC). Together, these findings provide novel information regarding the plasma lipoprotein changes and mutations in NPC1 disease, and suggest plasma HDL-C represents a potential biomarker of NPC1 disease severity. Niemann-Pick type C1 disease (NPC1) is an autosomal recessive lipid storage disorder characterized by clinical manifestations involving primarily the liver and brain (1Patterson M.C. Vanier M.T. Suzuki K. Morris J.E. Carstea E.D. Neufeld E.B. Blanchette-Mackie E.J. Pentchev P.G. Niemann-Pick Disease Type C: a lipid trafficking disorder.in: C. R. Beaudet A.L. Sly W.S. Valle D. The Metabolic and Molecular Bases of Inherited Disease. 8th edition. McGraw-Hill, New York2001: 3611-3633Google Scholar). The onset of signs or symptoms can occur at any age and have a variable phenotype. The classic clinical phenotype is also variable and includes mid-to-late childhood onset of gait disturbance followed by progressive neurodegeneration with vertical gaze palsy, seizures, and dementia, resulting in death during the second or third decade (2Vanier M.T. Wenger D.A. Comly M.E. Rousson R. Brady R.O. Pentchev P.G. Niemann-Pick disease group C: clinical variability and diagnosis based on defective cholesterol esterification. A collaborative study on 70 patients.Clin. Genet. 1988; 33: 331-348Google Scholar, 3Vanier M.T. Rodriguez-Lafrasse C. Rousson R. Gazzah N. Juge M.C. Pentchev P.G. Revol A. Louisot P. Type C Niemann-Pick disease: spectrum of phenotypic variation in disruption of intracellular LDL-derived cholesterol processing.Biochim. Biophys. Acta. 1991; 1096: 328-337Google Scholar, 4Vanier M.T. Rodriguez-Lafrasse C. Rousson R. Duthel S. Harzer K. Pentchev P.G. Revol A. Louisot P. Type C Niemann-Pick disease: biochemical aspects and phenotypic heterogeneity.Dev. Neurosci. 1991; 13: 307-314Google Scholar). The clinical phenotype of NPC1 disease has been categorized according to the age of onset of symptoms (5Fink J.K. Filling-Katz M.R. Sokol J. Cogan D.G. Pikus A. Sonies B. Soong B. Pentchev P.G. Comly M.E. Brady R.O. et al.Clinical spectrum of Niemann-Pick disease type C.Neurology. 1989; 39: 1040-1049Google Scholar, 6Omura K. Suzuki Y. Norose N. Sato M. Maruyama K. Koeda T. Type C Niemann-Pick disease: clinical and biochemical studies on 6 cases.Brain Dev. 1989; 11: 57-61Google Scholar), including an early-onset, rapidly progressive form associated with hepatic dysfunction and psychomotor delay during infancy, the classic form, and a late-onset type characterized by a slowly progressive intellectual impairment in adolescence or adulthood. The gene responsible for NPC1 disease, NPC1, was localized to chromosome 18 using linkage analysis and subsequently identified using positional mapping and molecular cloning techniques (7Carstea E.D. Polymeropoulos M.H. Parker C.C. Detera-Wadleigh S.D. O'Neill R.R. Patterson M.C. Goldin E. Xiao H. Straub R.E. Vanier M.T. et al.Linkage of Niemann-Pick disease type C to human chromosome 18.Proc. Natl. Acad. Sci. USA. 1993; 90: 2002-2004Google Scholar, 8Carstea E.D. Morris J.A. Coleman K.G. Loftus S.K. Zhang D. Cummings C. Gu J. Rosenfeld M.A. Pavan W.J. Krizman D.B. et al.Niemann-Pick C1 disease gene: homology to mediators of cholesterol homeostasis.Science. 1997; 277: 228-231Google Scholar). The encoded product, the Niemann-Pick C1 (NPC1) protein, contains several specialized regions (9Watari H. Blanchette-Mackie E.J. Dwyer N.K. Glick J.M. Patel S. Neufeld E.B. Brady R.O. Pentchev P.G. Strauss 3rd, J.F. Niemann-Pick C1 protein: obligatory roles for N-terminal domains and lysosomal targeting in cholesterol mobilization.Proc. Natl. Acad. Sci. USA. 1999; 96: 805-810Google Scholar, 10Watari H. Blanchette-Mackie E.J. Dwyer N.K. Watari M. Neufeld E.B. Patel S. Pentchev P.G. Strauss 3rd, J.F. Mutations in the leucine zipper motif and sterol-sensing domain inactivate the Niemann-Pick C1 glycoprotein.J. Biol. Chem. 1999; 274: 21861-21866Google Scholar, 11Watari H. Blanchette-Mackie E.J. Dwyer N.K. Watari M. Burd C.G. Patel S. Pentchev P.G. Strauss 3rd, J.F. Determinants of NPC1 expression and action: key promoter regions, posttranscriptional control, and the importance of a “cysteine-rich” loop.Exp. Cell Res. 2000; 259: 247-256Google Scholar), including a sterol-sensing domain also present in other key proteins regulating cholesterol metabolism, including 3-hydroxy-3-methyl-glutaryl-CoA reductase and sterol regulatory element-binding protein (SREBP) cleavage-activating protein (12Ohgami N. Ko D.C. Thomas M. Scott M.P. Chang C.C. Chang T.Y. Binding between the Niemann-Pick C1 protein and a photoactivatable cholesterol analog requires a functional sterol-sensing domain.Proc. Natl. Acad. Sci. USA. 2004; 101: 12473-12478Google Scholar, 13Liu R. Lu P. Chu J.W. Sharom F.J. Characterization of fluorescent sterol binding to purified human NPC1.J. Biol. Chem. 2009; 284: 1840-1852Google Scholar). Recent studies indicate cholesterol binds to luminal loop-1 of NPC1 (14Infante R.E. Radhakrishnan A. Abi-Mosleh L. Kinch L.N. Wang M.L. Grishin N.V. Goldstein J.L. Brown M.S. Purified NPC1 protein: II. Localization of sterol binding to a 240-amino acid soluble luminal loop.J. Biol. Chem. 2008; 283: 1064-1075Google Scholar), and that a separate N-terminal helical subdomain of NPC1 is required for cholesterol transfer between NPC2 and the cholesterol-binding domain of NPC1 (15Kwon H.J. Abi-Mosleh L. Wang M.L. Deisenhofer J. Goldstein J.L. Brown M.S. Infante R.E. Structure of N-terminal domain of NPC1 reveals distinct subdomains for binding and transfer of cholesterol.Cell. 2009; 137: 1213-1224Google Scholar). At the cellular level, decreased NPC1 protein function results in an accumulation of both cholesterol and glycosphingolipids within late endosomes and lysosomes (16Zervas M. Somers K.L. Thrall M.A. Walkley S.U. Critical role for glycosphingolipids in Niemann-Pick disease type C.Curr. Biol. 2001; 11: 1283-1287Google Scholar, 17te Vruchte D. Lloyd-Evans E. Veldman R.J. Neville D.C. Dwek R.A. Platt F.M. van Blitterswijk W.J. Sillence D.J. Accumulation of glycosphingolipids in Niemann-Pick C disease disrupts endosomal transport.J. Biol. Chem. 2004; 279: 26167-26175Google Scholar, 18Pentchev P.G. Comly M.E. Kruth H.S. Patel S. Proestel M. Weintroub H. The cholesterol storage disorder of the mutant BALB/c mouse. A primary genetic lesion closely linked to defective esterification of exogenously derived cholesterol and its relationship to human type C Niemann-Pick disease.J. Biol. Chem. 1986; 261: 2772-2777Google Scholar, 19Liscum L. Ruggiero R.M. Faust J.R. The intracellular transport of low density lipoprotein-derived cholesterol is defective in Niemann-Pick type C fibroblasts.J. Cell Biol. 1989; 108: 1625-1636Google Scholar). As a result, it is believed that the NPC1 protein has a central role in regulating the transport of these lipids out of late endosomes/lysosomes to other cellular compartments, including the Golgi apparatus, plasma membrane, and endoplasmic reticulum (20Blanchette-Mackie E.J. Dwyer N.K. Amende L.M. Kruth H.S. Butler J.D. Sokol J. Comly M.E. Vanier M.T. August J.T. Brady R.O. et al.Type-C Niemann-Pick disease: low density lipoprotein uptake is associated with premature cholesterol accumulation in the Golgi complex and excessive cholesterol storage in lysosomes.Proc. Natl. Acad. Sci. USA. 1988; 85: 8022-8026Google Scholar, 21Garver W.S. Krishnan K. Gallagos J.R. Michikawa M. Francis G.A. Heidenreich R.A. Niemann-Pick C1 protein regulates cholesterol transport to the trans-Golgi network and plasma membrane caveolae.J. Lipid Res. 2002; 43: 579-589Google Scholar, 22Puri V. Watanabe R. Dominguez M. Sun X. Wheatley C.L. Marks D.L. Pagano R.E. Cholesterol modulates membrane traffic along the endocytic pathway in sphingolipid-storage diseases.Nat. Cell Biol. 1999; 1: 386-388Google Scholar, 23Sagiv Y. Hudspeth K. Mattner J. Schrantz N. Stern R.K. Zhou D. Savage P.B. Teyton L. Bendelac A. Cutting edge: impaired glycosphingolipid trafficking and NKT cell development in mice lacking Niemann-Pick type C1 protein.J. Immunol. 2006; 177: 26-30Google Scholar). Consistent with this proposed function, studies performed in human and mouse fibroblasts have demonstrated that the NPC1 protein is primarily localized to a novel late endosome-like compartment capable of transiently interacting with LDL-derived cholesterol-enriched late endosomes/lysosomes (24Neufeld E.B. Wastney M. Patel S. Suresh S. Cooney A.M. Dwyer N.K. Roff C.F. Ohno K. Morris J.A. Carstea E.D. et al.The Niemann-Pick C1 protein resides in a vesicular compartment linked to retrograde transport of multiple lysosomal cargo.J. Biol. Chem. 1999; 274: 9627-9635Google Scholar, 25Garver W.S. Heidenreich R.A. Erickson R.P. Thomas M.A. Wilson J.M. Localization of the murine Niemann-Pick C1 protein to two distinct intracellular compartments.J. Lipid Res. 2000; 41: 673-687Google Scholar). Since the identification of the NPC1 gene, a number of studies have characterized various mutations and attempted to associate these mutations with a biochemical and clinical phenotype (26Greer W.L. Riddell D.C. Murty S. Gillan T.L. Girouard G.S. Sparrow S.M. Tatlidil C. Dobson M.J. Neumann P.E. Linkage disequilibrium mapping of the Nova Scotia variant of Niemann-Pick disease.Clin. Genet. 1999; 55: 248-255Google Scholar, 27Greer W.L. Dobson M.J. Girouard G.S. Byers D.M. Riddell D.C. Neumann P.E. Mutations in NPC1 highlight a conserved NPC1-specific cysteine-rich domain.Am. J. Hum. Genet. 1999; 65: 1252-1260Google Scholar, 28Meiner V. Shpitzen S. Mandel H. Klar A. Ben-Neriah Z. Zlotogora J. Sagi M. Lossos A. Bargal R. Sury V. et al.Clinical-biochemical correlation in molecularly characterized patients with Niemann-Pick type C.Genet. Med. 2001; 3: 343-348Google Scholar, 29Tarugi P. Ballarini G. Bembi B. Battisti C. Palmeri S. Panzani F. Di Leo E. Martini C. Federico A. Calandra S. Niemann-Pick type C disease: mutations of NPC1 gene and evidence of abnormal expression of some mutant alleles in fibroblasts.J. Lipid Res. 2002; 43: 1908-1919Google Scholar). To date, more than 243 different loss-of-function mutations of NPC1 have been reported (30Niemann-Pick Type C Disease Gene Variation Database. Available at http://npc.fzk.de/index.php?section=aboutGoogle Scholar, 31Park W.D. O'Brien J.F. Lundquist P.A. Kraft D.L. Vockley C.W. Karnes P.S. Patterson M.C. Snow K. Identification of 58 novel mutations in Niemann-Pick disease type C: correlation with biochemical phenotype and importance of PTC1-like domains in NPC1.Hum. Mutat. 2003; 22: 313-325Google Scholar), in addition to 60 different nondisease-causing polymorphisms (31Park W.D. O'Brien J.F. Lundquist P.A. Kraft D.L. Vockley C.W. Karnes P.S. Patterson M.C. Snow K. Identification of 58 novel mutations in Niemann-Pick disease type C: correlation with biochemical phenotype and importance of PTC1-like domains in NPC1.Hum. Mutat. 2003; 22: 313-325Google Scholar, 32Scott C. Ioannou Y.A. The NPC1 protein: structure implies function.Biochim. Biophys. Acta. 2004; 1685: 8-13Google Scholar, 33Fernandez-Valero E.M. Ballart A. Iturriaga C. Lluch M. Macias J. Vanier M.T. Pineda M. Coll M.J. Identification of 25 new mutations in 40 unrelated Spanish Niemann-Pick type C patients: genotype-phenotype correlations.Clin. Genet. 2005; 68: 245-254Google Scholar, 34Millat G. Marcais C. Rafi M.A. Yamamoto T. Morris J.A. Pentchev P.G. Ohno K. Wenger D.A. Vanier M.T. Niemann-Pick C1 disease: the I1061T substitution is a frequent mutant allele in patients of Western European descent and correlates with a classic juvenile phenotype.Am. J. Hum. Genet. 1999; 65: 1321-1329Google Scholar). Although the ability to establish meaningful genotype and clinical phenotype associations has been difficult, primarily due to the fact that most NPC1 patients are compound heterozygous for different mutations, some associations have been established for NPC1 patients with homozygous mutations. In particular, the relatively common I1061T mutation, present in approximately 20% of all known mutations for NPC1 and prominent among individuals of Western European descent, predisposes patients to the classic NPC1 clinical phenotype (34Millat G. Marcais C. Rafi M.A. Yamamoto T. Morris J.A. Pentchev P.G. Ohno K. Wenger D.A. Vanier M.T. Niemann-Pick C1 disease: the I1061T substitution is a frequent mutant allele in patients of Western European descent and correlates with a classic juvenile phenotype.Am. J. Hum. Genet. 1999; 65: 1321-1329Google Scholar, 35Yamamoto T. Ninomiya H. Matsumoto M. Ohta Y. Nanba E. Tsutsumi Y. Yamakawa K. Millat G. Vanier M.T. Pentchev P.G. et al.Genotype-phenotype relationship of Niemann-Pick disease type C: a possible correlation between clinical onset and levels of NPC1 protein in isolated skin fibroblasts.J. Med. Genet. 2000; 37: 707-712Google Scholar). Defects in cholesterol trafficking out of lysosomes might be expected to produce changes in plasma lipoprotein levels. We previously reported low plasma HDL cholesterol (HDL-C) levels in 17 of 21 NPC disease patients studied, and provided evidence using human NPC1−/− fibroblasts that this is due to defective upregulation of the key transporter mediating new HDL particle formation, ABCA1, as a consequence of impaired release of cholesterol from lysosomes (36Choi H.Y. Karten B. Chan T. Vance J.E. Greer W.L. Heidenreich R.A. Garver W.S. Francis G.A. Impaired ABCA1-dependent lipid efflux and hypoalphalipoproteinemia in human Niemann-Pick type C disease.J. Biol. Chem. 2003; 278: 32569-32577Google Scholar). We also found a tendency toward reduced plasma LDL cholesterol (LDL-C) and increased plasma triglycerides in these patients (36Choi H.Y. Karten B. Chan T. Vance J.E. Greer W.L. Heidenreich R.A. Garver W.S. Francis G.A. Impaired ABCA1-dependent lipid efflux and hypoalphalipoproteinemia in human Niemann-Pick type C disease.J. Biol. Chem. 2003; 278: 32569-32577Google Scholar). In the current study, we sought to determine whether these abnormalities are a consistent feature of NPC1 disease, and whether the reduction in HDL-C might be a reflection of the biochemical severity of the disease. Recently, two independent reports, including ours, have been published providing an in depth analysis of the natural history, clinical features, and disabilities associated with NPC1 disease (37Imrie J. Dasgupta S. Besley G.T. Harris C. Heptinstall L. Knight S. Vanier M.T. Fensom A.H. Ward C. Jacklin E. et al.The natural history of Niemann-Pick disease type C in the UK.J. Inherit. Metab. Dis. 2007; 30: 51-59Google Scholar, 38Garver W.S. Francis G.A. Jelinek D. Shepherd G. Flynn J. Castro G. Walsh Vockley C. Coppock D.L. Pettit K.M. Heidenreich R.A. et al.The National Niemann-Pick C1 disease database: report of clinical features and health problems.Am. J. Med. Genet. A. 2007; 143A: 1204-1211Google Scholar). These reports confirm the heterogeneous nature of NPC1 disease, and have identified particular symptoms including cataplexy and epilepsy being more common than previously reported. The present analysis of the National NPC1 Disease Database reports i) further characterization of plasma lipoprotein abnormalities in NPC1 disease, ii) associations between the lipid profiles, gene mutations, and biochemical phenotypes in NPC1 patients, iii) possible correlations between the lipid profiles, age of diagnosis and death, and the average abilities for NPC1 patients, and iv) the NPC1 mutations including five novel mutations present in the database cohort. The development and initial information obtained for the National NPC1 Disease Database was provided in an earlier report (38Garver W.S. Francis G.A. Jelinek D. Shepherd G. Flynn J. Castro G. Walsh Vockley C. Coppock D.L. Pettit K.M. Heidenreich R.A. et al.The National Niemann-Pick C1 disease database: report of clinical features and health problems.Am. J. Med. Genet. A. 2007; 143A: 1204-1211Google Scholar). The plan to establish a National NPC1 Disease Database was introduced during the annual National Niemann-Pick Disease Foundation Family Conference, sponsored in part by the Ara Parseghian Medical Research Foundation (APMRF), in 2003. Those families expressing an interest in such a study were asked to provide their name, home address, and phone number for later contact by the APMRF. The study design was reviewed and approved by the University of Arizona Institutional Review Board through the Human Subjects Protection Program. The families were then mailed the NPC1 disease clinical questionnaire, in addition to a parental consent form, a subject's consent form, and a minor's assent form, each of which required the proper signatures and dates for the information to be entered into the database and used for the present study. An NPC1 disease clinical questionnaire, previously prepared and graciously provided by Dr. Mercè Pineda (Hospital Sant Joan de Déu, Barcelona), was translated into English and modified for use in creating an American National NPC1 Disease Database. The questionnaire consisted of 83 questions, including one for the specific gene mutations and four for the lipid profile components (total cholesterol, HDL-C, LDL-C, and triglycerides). The study was conducted over a one-year period from December 2003 to December 2004. The questionnaire was completed by parents/caregivers and/or physicians responsible for patients with NPC1 disease living in the United States. The questionnaire did not specifically request information concerning who provided the initial diagnosis of NPC1 disease (primary care physician, pediatrician, neurologist, or other specialist). However, in all cases the parents/caregivers and/or patients were informed of the diagnosis for NPC1 disease after either genotype analysis, enhanced filipin staining, and/or decreased cholesterol esterification using cultured fibroblasts derived from patients, consistent with the age at diagnosis reported in the questionnaire and the time of identification of the NPC1 gene. Fasting lipid profiles, all of which were obtained from a certified clinical diagnostic laboratory, were either mailed or faxed from parents/caregivers or their physicians responsible for the patient with NPC1 disease. Components of the lipid profile obtained from patients in the NPC1 Database were compared with normal ranges for children between 5 and 9 years of age and adults between 25 and 29 years of age of both genders, representing the average range of ages for children and adults in the NPC1 Disease Database, using The Lipid Research Clinics Population Studies Data Book published by the National Institutes of Health (39Lipid Metabolism Branch, National Heart, Lung, and Blood Institute, National Institutes of Health The Lipid Research Clinics Population Studies Data Book, The prevalence study, NIC Publication No. 79–1527 ed. US Department of Health and Human Services, Public Health Service, Bethesda, MD1980Google Scholar). NPC1 gene mutations were determined at the Mayo Clinic (Rochester, MN) and provided to the APMRF. Through written parental or patient consent, these mutations were then made available for the National NPC1 Disease Database. In some cases, amino acid but not nucleotide changes were provided. The majority of known NPC1 mutations have been associated with specific biochemical phenotypes as defined by measuring the esterification of LDL-derived cholesterol in skin fibroblasts obtained from NPC1 patients (28Meiner V. Shpitzen S. Mandel H. Klar A. Ben-Neriah Z. Zlotogora J. Sagi M. Lossos A. Bargal R. Sury V. et al.Clinical-biochemical correlation in molecularly characterized patients with Niemann-Pick type C.Genet. Med. 2001; 3: 343-348Google Scholar, 31Park W.D. O'Brien J.F. Lundquist P.A. Kraft D.L. Vockley C.W. Karnes P.S. Patterson M.C. Snow K. Identification of 58 novel mutations in Niemann-Pick disease type C: correlation with biochemical phenotype and importance of PTC1-like domains in NPC1.Hum. Mutat. 2003; 22: 313-325Google Scholar, 34Millat G. Marcais C. Rafi M.A. Yamamoto T. Morris J.A. Pentchev P.G. Ohno K. Wenger D.A. Vanier M.T. Niemann-Pick C1 disease: the I1061T substitution is a frequent mutant allele in patients of Western European descent and correlates with a classic juvenile phenotype.Am. J. Hum. Genet. 1999; 65: 1321-1329Google Scholar, 40Millat G. Marcais C. Tomasetto C. Chikh K. Fensom A.H. Harzer K. Wenger D.A. Ohno K. Vanier M.T. 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.Am. J. Hum. Genet. 2001; 68: 1373-1385Google Scholar, 41Bauer P. Knoblich R. Bauer C. Finckh U. Hufen A. Kropp J. Braun S. Kustermann-Kuhn B. Schmidt D. Harzer K. et al.NPC1: complete genomic sequence, mutation analysis, and characterization of haplotypes.Hum. Mutat. 2002; 19: 30-38Google Scholar). To further analyze NPC1 mutations and biochemical phenotypes in relation to different components of the lipid profile, the biochemical phenotypes were assigned a severity score based on the reduction in cholesterol esterification by ACAT as previously described (31Park W.D. O'Brien J.F. Lundquist P.A. Kraft D.L. Vockley C.W. Karnes P.S. Patterson M.C. Snow K. Identification of 58 novel mutations in Niemann-Pick disease type C: correlation with biochemical phenotype and importance of PTC1-like domains in NPC1.Hum. Mutat. 2003; 22: 313-325Google Scholar). For example, a variant biochemical phenotype, represented by a mild mutation with a near normal biochemical phenotype, was given a score of one. A moderate biochemical phenotype, represented by an intermediate mutation and biochemical phenotype, was given a score of two. A classical biochemical phenotype, represented by a classical mutation that was more severe than the moderate mutation but less severe than a severe mutation, was given a score of three. A severe biochemical phenotype, represented by a severe mutation and biochemical phenotype, was given a score of four. In cases where the biochemical phenotype was unknown, no score was assigned (unknown = U). In order to assign a biochemical phenotype score for the combination of two different NPC1 mutations, the following rules were applied: 1) One moderate mutation always conferred a moderate biochemical phenotype, 2) classical mutations known to be variable always depended upon the other mutation, and 3) one severe mutation always conferred a severe biochemical phenotype. The clinical information, lipid profiles, and gene mutations were entered into a Microsoft ACCESS database and analyzed by the Data Analysis Unit, University of Arizona Program Site of the Arizona University Center on Disabilities. Pearson correlation coefficients were computed to evaluate relationships among most of the variables in this report. However, Spearman's rank correlation coefficient was substituted whenever the biochemical phenotype score, which represents an ordinal scale variable, was used in an analysis. Significance was determined by P-values < 0.05. All statistics and significance tests were computed using SPSS version 14.0. For the present study, a total of 136 questionnaires were mailed to NPC1 families living in the United States with 88 of these questionnaires (65%) being returned. For the 88 questionnaires returned, the lipid profiles from 34 patients (39%) were made available. The patients in this sample were represented by an approximately equal number of males (N = 18) and females (N = 16), ranging in age between 1.5 and 45 years and at different stages and severity of disease. For these patients, the average concentrations (± SE) of total cholesterol were 146 ± 5.6 mg/dl (3.78 ± 0.14 mmol/l), HDL-C 35 ± 2.0 mg/dl (0.91 ± 0.05 mmol/l), LDL-C 86 ± 5.0 mg/dl (2.22 ± 0.13 mmol/l), and triglycerides 126 ± 8.7 mg/dl (1.42 ± 0.10 mmol/l). The lipid profiles were grouped by gender and age ≤14 or >14 to determine whether there were gender or age-specific differences among these patients (Fig. 1). No significant differences were found among any of the lipid parameters when comparing male or female NPC1 patients based on age <14 or >14. However, several significant differences were found when comparing the NPC1 patient lipid levels with a control population represented by normal males and females 5–9 and 25–29 years of age as reported in the Lipid Research Clinics Population Studies Data Book (39Lipid Metabolism Branch, National Heart, Lung, and Blood Institute, National Institutes of Health The Lipid Research Clinics Population Studies Data Book, The prevalence study, NIC Publication No. 79–1527 ed. US Department of Health and Human Services, Public Health Service, Bethesda, MD1980Google Scholar) (Table 1). Total cholesterol levels were found to be significantly lower in female children and adult men with NPC1 disease but not in male children or adult females. LDL-C was significantly lower than controls in female children and adult males and females with NPC1 disease but not male children. Triglycerides were found to be significantly higher in male and female children and adult women with NPC1 disease but not adult males. HDL-C was found to be significantly lower in all male and female NPC1 patient groups when compared with controls, regardless of age, with HDL-C ranging from 56% to 78% lower than controls across the groups. In comparison with the lipid levels reported in our initial study in 2003 (36Choi H.Y. Karten B. Chan T. Vance J.E. Greer W.L. Heidenreich R.A. Garver W.S. Francis G.A. Impaired ABCA1-dependent lipid efflux and hypoalphalipoproteinemia in human Niemann-Pick type C disease.J. Biol. Chem. 2003; 278: 32569-32577Google Scholar), where 17 of 21 patients (81%) had an HDL-C level below 40 mg/dl or 1.03 mmol/l (all of whom are included in the current analysis), the present study group contained 28 of 34 subjects (82%) with HDL-C levels below this cutoff. Using currently accepted lower limits of HDL-C for adults based on the National Cholesterol Education Program ATP III guidelines definition of the metabolic syndrome (<40 mg/dl or 1.0 mmol/l for men and <50 mg/dl or 1.29 mmol/l for women) (42Grundy S.M. Brewer Jr., H.B. Cleeman J.I. Smith Jr., S.C. Lenfant C. Definition of metabolic syndrome: report of the National Heart, Lung, and Blood Institute/American Heart Association conference on scientific issues related to definition.Circulation. 2004; 109: 433-438Google Scholar), 14 of 18 or 78% of NPC1 male patients and 15 of 16 or 94% of female NPC1 patients had HDL-C levels below these norms.TABLE 1Average concentration of total cholesterol, HDL cholesterol, LDL cholesterol, and triglycerides for normal individuals and NPC1 patients based on both gender and ageTotal CholesterolHDL CholesterolLDL CholesterolTriglyceridesNormal Males159.9 ± 0.755.8 ± 1.092.5 ± 1.855.7 ± 0.6Age 5–9 Years4.13 ± 0.01.44 ± 0.02.39 ± 0.00.63 ± 0.0N = 1253N = 145N = 132N = 1253NPC1 Males149.3 ± 15.034.4 ± 7.186.9 ± 15.0136.3 ± 21.6Age 7.9 ± 1.4 Years3.86 ± 0.40.89 ± 0.82.25 ± 0.41.54 ± 0.2N = 7N = 7N = 7N = 7SignificanceNSP = 0.02NSP = 0.01Normal Females163.7 ± 0.753.2 ± 1.0100.4 ± 2.160.3 ± 0.8Age 5–9 Years4.23 ± 0.01.38 ± 0.02.60 ± 0.070.68 ± 0.0N = 1118N = 127N = 114N = 1118NPC1 Females124.6 ± 7.030.3 ± 3.075.0 ± 6.799.3 ± 9.5Age 9.9 ± 0.9 Y
Niemann-Pick type C1 (NPC1) disease is an autosomal-recessive cholesterol-storage disorder characterized by liver dysfunction, hepatosplenomegaly, and progressive neurodegeneration. The NPC1 gene is expressed in every tissue of the body, with liver expressing the highest amounts of NPC1 mRNA and protein. A number of studies have now indicated that the NPC1 protein regulates the transport of cholesterol from late endosomes/lysosomes to other cellular compartments involved in maintaining intracellular cholesterol homeostasis. The present study characterizes liver disease and lipid metabolism in NPC1 mice at 35 days of age before the development of weight loss and neurological symptoms. At this age, homozygous affected (NPC1(-/-)) mice were characterized with mild hepatomegaly, an elevation of liver enzymes, and an accumulation of liver cholesterol approximately four times that measured in normal (NPC1(+/+)) mice. In contrast, heterozygous (NPC1(+/-)) mice were without hepatomegaly and an elevation of liver enzymes, but the livers had a significant accumulation of triacylglycerol. With respect to apolipoprotein and lipoprotein metabolism, the results indicated only minor alterations in NPC1(-/-) mouse serum. Finally, compared to NPC1(+/+) mouse livers, the amount and processing of SREBP-1 and -2 proteins were significantly increased in NPC1(-/-) mouse livers, suggesting a relative deficiency of cholesterol at the metabolically active pool of cholesterol located at the endoplasmic reticulum. The results from this study further support the hypothesis that an accumulation of lipoprotein-derived cholesterol within late endosomes/lysosomes, in addition to altered intracellular cholesterol homeostasis, has a key role in the biochemical and cellular pathophysiology associated with NPC1 liver disease.
