Cholesterol signaling at the endoplasmic reticulum occurs in npc1(-/-) but not in npc1(-/-), LDLR(-/-) mice.

It remains controversial whether deficiency of the Niemann-Pick C1 (npc1) protein results in altered cholesterol signaling at the endoplasmic reticulum (ER). In this report, we have measured the processed, nuclear form of sterol regulatory element binding protein (SREBP)-1 in livers of npc1 wild-type, heterozygous, and homozygous deficient mice, alone, and in combination with deficiencies of the low density lipoprotein receptor (LDLR) or the multiple drug resistant (mdr)1a, P-glycoprotein. Cleavage of SREBPs to activated forms normally occurs when the ER is deficient in cholesterol. A large decrease in processed SREBP-1 was evident in fasted npc1(-/-) mice and npc1(-/-), mdr1a(-/-) mice, with no decrease evident in npc1(-/-), LDLR(-/-) mice. These results suggest that the increase in cellular cholesterol which occurs in npc1(-/-) and in npc1(-/-), mdr1a(-/-) mice includes the sites responsible for cholesterol signaling, while the similar increase in cholesterol found in npc1(-/-), LDLR(-/-) mice does not.

Site-specific phosphorylation of tau accompanied by activation of mitogen-activated protein kinase (MAPK) in brains of Niemann-Pick type C mice.

Niemann-Pick type C (NPC) disease is characterized by an accumulation of cholesterol in most tissues and progressive neurodegeneration with the formation of neurofibrillary tangles. Neurofibrillary tangles are composed of paired helical filaments (PHF), a major component of which is the hyperphosphorylated tau. In this study we used NPC heterozygous and NPC homozygous mouse brains to investigate the molecular mechanism responsible for tauopathy in NPC. Immunoblot analysis using anti-tau antibodies (Tau-1, PHF-1, AT-180, and AT-100) revealed site-specific phosphorylation of tau at Ser-396 and Ser-404 in the brains of NPC homozygous mice. Mitogen-activated protein kinase, a potential serine kinase known to phosphorylate tau, was activated, whereas other serine kinases such as glycogen synthase kinase-3beta and cyclin-dependent kinase 5 were inactive. Morphological examination demonstrated that a number of neurons, the perikarya of which strongly immunostained with PHF-1, exhibited polymorphorous cytoplasmic inclusion bodies and multi-concentric lamellar-like bodies. Importantly, the accumulation of intracellular cholesterol in NPC mouse brains was determined to be a function of age. From these results we conclude that abnormal cholesterol metabolism due to the genetic mutation in NPC1 may be responsible for activation of the mitogen-activated protein kinase-signaling pathway and site-specific phosphorylation of tau in vivo, leading to tauopathy in NPC.

Cyclodextrins in the treatment of a mouse model of Niemann-Pick C disease.

Niemann-Pick type C (NPC) is a neurodegenerative disorder characterized by greatly altered somatic cholesterol metabolism. The NPC1 gene has recently been cloned and shown to have sequence homology to other sterol-sensing proteins. We have used a mouse model with a disrupted npc1 gene to study the effects of the cholesterol-mobilizing compound, 2-hydroxypropyl-beta-cyclodextrins (HPBCD), on the clinical course of this disorder. Treatment with two HPBCDs, with varying levels of 2-hydroxypropyl substitution, had effects in delaying neurological symptoms and in decreasing liver cholesterol storage while a third HPBCD was without effect. The ameliorating effect was not improved by longer exposure times (commencement of exposure in utero), however, it is not known if there is transplacental transfer of HPBCDs. The combination of HPBCD with probucol or nifedipine (which have previously been shown to lower liver cholesterol in this animal model) markedly decreased liver storage of unesterified cholesterol without altering the depressed levels of esterified cholesterol. The slight effects of the HPBCDs on neurological symptoms may be partially due to their apparent non-permeation of the blood-brain barrier (BBB). This non-permeation was assayed with radioactive tracers and was also present in the mdr1a knockout mice which have greatly increased BBB permeability for many drugs. Intrathecal delivery of HPBCD by an Alzet osmotic minipump did not improve its efficacy in ameliorating neurological symptoms.

Variable presentation of Rothmund-Thomson syndrome.

The recent finding that a subset of patients with Rothmund-Thomson syndrome (RTS) have mutations of a helicase gene has prompted reexamination of the phenotypes of individuals diagnosed with this disorder. We report on two patients with variable presentations of RTS. Initial presenting symptoms included growth deficiency and absent thumbs in one patient and osteogenic sarcoma and poikiloderma in the second patient. The growth-deficient patient was diagnosed with growth hormone deficiency and had a subnormal response to growth hormone supplementation. Neither malformations nor growth deficiency were present in the patient with osteogenic sarcoma, and her only other manifestation of RTS was poikiloderma. The diagnosis of RTS should be considered in all patients with osteogenic sarcoma, particularly if associated with skin changes.

Tumor endothelium-specific transgene expression directed by vascular endothelial growth factor receptor-2 (Flk-1) promoter/enhancer sequences.

The receptor tyrosine kinase Flk-1 plays a pivotal role in the development of the vascular system and in the vascularization of a wide variety of tumors. We have investigated the activity of cis-acting sequences of the murine Flk-1 gene in the tumor endothelium of experimental tumor models in vivo. B16 melanoma, BFS-1 fibrosarcoma, and polyoma middle T-induced mammary adenocarcinoma were grown in transgenic mice that express the LacZ reporter gene under the control of a 939-bp Flk-1 promoter fragment and an enhancer element located in a 2.3-kb fragment of the first intron. In all experimental tumor models examined, strong endothelium-specific reporter gene expression was observed while being absent from most blood vessels in normal adult tissue. The expression patterns of the LacZ reporter gene correlate well between established tumors grown in Flk1-LacZ transgenic mice and tumors grown in Flk-1+/LacZ knock-in mice that express the LacZ reporter gene from the endogenous Flk-1 locus. The endothelium-specific activity of the Flk-1 promoter/enhancer sequences in three different experimental tumor models demonstrates that the regulatory sequences that mediate the up-regulation of Flk-1 in the tumor endothelium are contained in the Flk-1 promoter/enhancer sequences used, and that these elements function relatively independently of the tumor type. The Flk-1 promoter/enhancer sequences should allow the analysis of the signaling pathways that lead to the up-regulation of Flk-1 in the tumor endothelium and to specifically target therapeutic genes to the endothelium of tumors for antiangiogenic tumor therapy.

De novo expression of vascular endothelial growth factor in human pancreatic cancer: evidence for an autocrine mitogenic loop.

BACKGROUND & AIMS: The role of vascular endothelial growth factor (VEGF) and its receptors in tumor angiogenesis has been well established. We analyzed the expression pattern and biologic significance of VEGF and its receptors in human pancreatic cancer. METHODS: VEGF, KDR/flk-1, and flt-1 expression were examined by immunohistochemistry, in situ hybridization, reverse-transcription polymerase chain reaction, enzyme-linked immunosorbent assay, and receptor phosphorylation. VEGF-stimulated mitogenesis was investigated by mitogen-activated protein kinase (MAPK) phosphorylation, transactivation of a c-fos promoter reporter construct, DNA synthesis assays, and stable transfection of a dominant-negative flk-1 complementary DNA (cDNA) construct. RESULTS: Compared with normal pancreas and chronic pancreatitis, VEGF and its receptors were overexpressed in pancreatic cancer. KDR and flt-1 were detected not only in endothelial cells but also in tumor cells. VEGF expression was observed in all human pancreatic tumor cell lines examined, and the KDR/flk-1 and flt-1 receptor was detected in 2 cell lines. VEGF treatment results in phosphorylation of MAPKs, transactivation of a c-fos promoter construct, and growth stimulation in KDR/flk-1-expressing cell lines, which could be blocked by VEGF antagonists. Furthermore, stable transfection of a dominant-negative flk-1 cDNA significantly inhibited tumor cell growth. CONCLUSIONS: These results not only support the important role of the VEGF/VEGF receptor system in pancreatic tumor biology but also suggest the existence of an autocrine/paracrine mitogenic loop for pancreatic cancer cells.

Fine linkage and physical mapping suggests cross-over suppression with a retroposon insertion at the npc1 mutation.

Mouse Niemann-Pick disease type C1 (npc1), formerly designated spm (sphingomyelinosis), is an autosomal recessive lipid storage disorder. We generated a high-resolution linkage map in the 2.24-cM npc1 critical region by typing eight polymorphic markers in 2322 meioses (948 of these were previously reported). A minimal set of overlapping yeast artificial chromosomes (YACs) had previously been assembled (Hsu and Erickson 2000). The YAC 313-B-8, which covered this whole region, has been used to construct cosmid libraries. Three cosmid contigs were built, and one of them contained the npc1 locus. Two (CA)(n) microsatellites were identified, and the one new one was characterized, from the YAC-derived cosmids. The most proximal cosmid contig overlaps with markers near twirler (Tw). Both the physical map and genetic linkage map have been integrated to study the recombination frequencies in this particular region of the mouse genome, and recombination suppression due to the heterozygous insertion of DNA was suggested.

Fumaric aciduria: clinical and imaging features.

Fumaric aciduria (fumaric acidemia, fumarase deficiency) is a rare inborn error of metabolism caused by deficient activity of fumarate hydratase, one of the constituent enzymes of the Krebs tricarboxylic acid cycle. We describe the clinical and imaging features of this disease arising from a consanguineous pedigree in 8 patients in the southwestern United States. Thirteen patients have been previously described in the medical literature. Our patients presented with an early infantile encephalopathy with profound developmental retardation and hypotonia, and most experienced seizures. Previously unreported characteristics described here include structural brain malformations, dysmorphic facial features, and neonatal polycythemia. Magnetic resonance imaging showed multiple abnormalities, including diffuse polymicrogyria, decreased cerebral white matter, large ventricles, and open opercula. Fumaric aciduria should be included in the differential diagnosis of inborn errors of metabolism that cause cerebral malformations and dysmorphic features. The possibility that inborn errors of energy metabolism may cause structural malformations deserves increased recognition.

Localization of the murine Niemann-Pick C1 protein to two distinct intracellular compartments.

Niemann-Pick type C (NPC) disease is characterized by an accumulation of cholesterol and other lipids in the lysosomal compartment. In this report, we use subcellular fractionation and microscopy to determine the localization of the murine Niemann-Pick C1 (NPC1) protein. Fractionation of mouse liver homogenates indicates that some NPC1 cosediments with lysosome-associated membrane protein 1 (LAMP1)-containing membranes. However, a significant amount of NPC1 is also found in membranes that do not contain LAMP1. Moreover, fractionation of liver membranes and fibroblasts in the presence of a nonionic detergent showed that a fraction of NPC1 cosediments with caveolin-1 in rafts. Immunofluorescence microscopy of cultured mouse fibroblasts showed that NPC1 is found in two morphologically distinct structures. The first population is characterized by large punctate structures that do not colocalize with major organelle protein markers, but do colocalize with filipin and a small fraction of caveolin-1. Examination of these large NPC1-containing compartments using electron microscopy shows that these structures contain extensive internal membranes. The second population is represented by smaller, more diffuse structures, a fraction of which colocalize with LAMP1-positive compartments. Incubation of fibroblasts with low density lipoprotein (LDL) increases colocalization of NPC1 with LAMP1-containing compartments. This colocalization can be further enhanced by treating fibroblasts with progesterone or chloroquine. The results indicate that NPC1 is associated with an unique vesicular compartment enriched with cholesterol and containing caveolin-1, and that NPC1 cycles to LAMP1-positive compartments, presumably to facilitate the processing of LDL-derived cholesterol.

Pharmacological and genetic modifications of somatic cholesterol do not substantially alter the course of CNS disease in Niemann-Pick C mice.

Niemann-Pick type C (NPC) is a neurodegenerative disorder with somatically altered cholesterol metabolism. The NPC1 gene has recently been cloned and shown to have sequences shared with known sterol-sensing proteins. We have used a mouse model with a disrupted Npc1 gene to study two cholesterol-lowering drugs (nifedipine and probucol) and the effects of introducing a null mutation in the low-density lipoprotein receptor (LDLR). Although these treatments significantly ameliorated liver cholesterol storage, little effect on the onset of neurological symptoms was found.

The Npc1 mutation causes an altered expression of caveolin-1, annexin II and protein kinases and phosphorylation of caveolin-1 and annexin II in murine livers.

We have previously demonstrated (1) an increased expression of caveolin-1 in murine heterozygous and homozygous Niemann-Pick type C (NPC) livers, and (2) an increased concentration of unesterified cholesterol in a detergent insoluble caveolae-enriched fraction from homozygous livers. To define further the relationship between caveolin-1 function and the cholesterol trafficking defect in NPC, we examined the expression and distribution of additional caveolar and signal transduction proteins. The expression of annexin II was significantly increased in homozygous liver homogenates and the Triton X-100 insoluble floating fraction (TIFF). Phosphoamino acid analysis of caveolin-1 and annexin II from the homozygous TIFF demonstrated an increase in serine and tyrosine phosphorylation, respectively. To determine the basis for increased phosphorylation of these proteins, the expression and distribution of several protein kinases was examined. The expression of PKCalpha, PKCzeta and pp60-src (protein kinases) were significantly increased in both heterozygous and homozygous liver homogenates, while PKCdelta was increased only in homozygous livers. Of the protein kinases analyzed, only CK IIalpha was significantly enriched in the heterozygous TIFF. Finally, the concentration of diacylglycerol in the homozygous TIFF was significantly increased and this elevation may modulate PKC distribution and function. These results provide additional evidence for involvement of a caveolin-1 containing cellular fraction in the pathophysiology of NPC and also suggest that the Npc1 gene product may directly or indirectly, regulate the expression and distribution of signaling molecules.

Mutations in the pterin-4alpha-carbinolamine dehydratase (PCBD) gene cause a benign form of hyperphenylalaninemia.

Four patients with primapterinuria, postulated to be due to pterin-4alpha-carbinolamine dehydratase (PCD) deficiency, were diagnosed by biochemical and DNA analysis. All four patients presented in the neonatal period with hyperphenylalaninemia, and elevated neopterin and decreased biopterin levels in the urine. These symptoms are common to 6-pyruvoyltetrahydropterin synthase deficiency and thus there is a danger of misdiagnosis. In addition, all four patients had elevated urinary excretion of primapterin (7-biopterin), the only persistent biochemical abnormality. Analysis of fibroblast DNA from the patients identified the following mutations in the PCBD gene: one patient homozygous for the missense mutation E96K and one homozygous for the nonsense mutation Q97X, both in exon 4; one compound heterozygote with the mutations E96K and Q97X; and one patient with two different homozygous mutations: E26X in exon 2 and R87Q in exon 4. In two families, the parents were investigated and found to be obligate heterozygotes for particular mutations. One sibling was found to be unaffected. These results further substantiate the idea that primapterinuria is associated with mutations in the PCBD gene.

Altered expression of caveolin-1 and increased cholesterol in detergent insoluble membrane fractions from liver in mice with Niemann-Pick disease type C.

Niemann-Pick type C (NPC) is an autosomal recessive disease characterized by impaired cholesterol homeostasis due to a defect in the intracellular transport of unesterified cholesterol. While accumulation of lysosomal cholesterol is the most apparent microscopic finding, cholesterol has also been shown to accumulate in the trans-cisternae of the Golgi apparatus. Caveolin-1, a cholesterol-binding protein that cycles between the Golgi apparatus and the plasma membrane, has been hypothesized to participate in cholesterol transport. Using the BALB/c murine model for NPC disease, we found that the expression of caveolin-1 in total liver homogenates from heterozygous and homozygous affected animals was altered. Immunoblot analysis of liver homogenates from heterozygous mice revealed that caveolin-1 is significantly (p < 0.005) elevated, 4.9 fold, compared to normal mice. Total liver homogenates from homozygous affected mice also had a significant (p < 0.05) increase in caveolin-1 expression, 1.6 fold, compared to normal mice. Immunohistochemical staining of liver cross-sections revealed that the increased caveolin-1 was localized to sinusoidal endothelial cells in heterozygous mice. The Triton insoluble floating fraction (TIFF) was isolated using liver from each genotype and analyzed for caveolin-1 expression. Caveolin-1 in the TIFF from heterozygous mice was significantly (p < 0.01) elevated, 1.8 fold, compared to normal and homozygous affected animals; normal and homozygous affected animals, however, were not significantly different from each other. The TIFF isolated from homozygous affected mice revealed a 15 fold increase in unesterified cholesterol compared to the TIFF isolated from heterozygous and normal mice. In summary, these findings demonstrate an altered expression of caveolin-1 in liver from heterozygous and homozygous NPC mice and increased concentration of cholesterol from TIFF in homozygous affected NPC mice. The identification of these alterations in the TIFF suggests involvement of detergent insoluble membrane structures, possibly caveolae and/or detergent insoluble glycosphingolipid-enriched complexes (DIGs), in the cholesterol trafficking defect in this disorder.

Increased expression of caveolin-1 in heterozygous Niemann-Pick type II human fibroblasts.

Human Niemann-Pick type II fibroblasts, which encompass the panethnic type C (NPC) and Nova Scotia Acadian type D (NPD) variants, exhibit altered expression of caveolin-1 protein when examined by immunoblotting using an anti-caveolin-1 monoclonal antibody. Unexpectedly, caveolin-1 in heterozygous fibroblasts was significantly elevated as much as 10-fold compared to caveolin-1 in normal and homozygous affected fibroblasts. Homozygous NPC fibroblasts expressed caveolin-1 levels similar to those in normal fibroblasts, while the expression of caveolin-1 in homozygous NPD fibroblasts was slightly elevated. Northern analysis indicates that normal fibroblasts and NPC heterozygous fibroblasts have similar amounts of caveolin-1 mRNA, while NPC homozygous fibroblasts have significantly less caveolin-1 mRNA. In contrast, heterozygous and homozygous NPD fibroblasts exhibit increased levels of caveolin-1 mRNA. These novel findings suggest that caveolin-1 containing subcellular structures are involved in the pathophysiology of Niemann-Pick type II disease. Furthermore, altered caveolin-1 protein expression may serve as a useful marker for the diagnosis of carriers of NPC or NPD.

Guidelines for the retention, storage, and use of residual dried blood spot samples after newborn screening analysis: statement of the Council of Regional Networks for Genetic Services.

These guidelines provide scientific information for policy development by state health departments considering appropriate use of newborn screening specimens after screening tests are finished. Information was collected, debated, and formulated into a policy statement by the Newborn Screening Committee of the Council of Regional Networks for Genetic Services (CORN), a federally funded national consortium of representatives from 10 regional genetics networks. Newborn screening programs vary widely in approaches and policies concerning residual dried blood spot samples (DBS) collected for newborn screening. Recognition of the epidemiological utility of DBS samples for HIV seroprevalence surveys and a growing interest in DBSs for DNA analysis has intensified consideration of issues regarding retention, storage, and use of residual DBS samples. Potentially these samples provide a genetic material "bank" for all newborns nationwide. Their values as a resource for other uses has already been recognized by scientists, administrators, and judicial officials. Programs should promulgate rules for retention and use of residual newborn screening DBS samples based on scientifically valid information. Banking of newborn samples as sources of genetic material should be considered in light of potential benefit or harm to society.

Distal arthrogryposis type IIB: further clinical delineation and 54-year follow-up of an index case.

Distal arthrogryposis IIB is characterized by contractures of the distal joints (especially of the fingers and toes) and ptosis. We recently encountered a father and son with these manifestations. The father was reported 54 years ago as a case of amyoplasia congenita (arthrogryposis multiplex congenita). Both father and son have distal joint contractures, most severe in the hands and feet, as well as ptosis and ophthalmoplegia. In addition, these patients have an unusual distribution of hair loss, and conical teeth. Whether these latter findings are related to the type of distal arthrogryposis present in this family is not known. In spite of their physical limitations both father and son have maintained an active life-style.

A missense mutation (I278T) in the cystathionine beta-synthase gene prevalent in pyridoxine-responsive homocystinuria and associated with mild clinical phenotype.

Cystathionine beta-synthase (CBS) deficiency is an autosomal recessive disorder characterized by homocystinuria and multisystem clinical disease. Patients responsive to pyridoxine usually have a milder clinical phenotype than do nonresponsive patients, and we studied the molecular pathology of this disorder in an attempt to understand the molecular basis of the clinical variation. We previously reported a T833C transition in exon 8 causing a substitution of threonine for isoleucine at codon 278 (I278T). By PCR amplification and sequencing of exon 8 from genomic DNA we have now detected the I278T mutation in 7 of 11 patients with in vivo pyridoxine responsiveness and in 0 of 27 pyridoxine-nonresponsive patients. Two pyridoxine-responsive patients are homozygous and five are heterozygous for I278T. We have now observed the I278T mutation in 41% (9 of 22) of the independent alleles in pyridoxine-responsive patients of varied ethnic backgrounds. In two of the compound heterozygotes we identified a novel mutation (G139R and E144K) in the other allele. The finding that the two patients who are homozygous for I278T have only ectopia lentis and mild bone demineralization suggests that this mutation is associated with both in vivo pyridoxine responsiveness and mild clinical disease. Compound heterozygous patients who have one copy of this missense mutation are likely to retain some degree of pyridoxine responsiveness.