CTNS mutations in an American-based population of cystinosis patients

Nephropathic cystinosis is an autosomal recessive lysosomal storage disease characterized by renal failure at 10 years of age and other systemic complications. The gene for cystinosis, CTNS, has 12 exons. Its 2.6-kb mRNA codes for a 367-amino-acid putative cystine transporter with seven transmembrane domains. Previously reported mutations include a 65-kb "European" deletion involving marker D17S829 and 11 small mutations. Mutation analysis of 108 American-based nephropathic cystinosis patients revealed that 48 patients (44%) were homozygous for the 65-kb deletion, 2 had a smaller major deletion, 11 were homozygous and 3 were heterozygous for 753G-->A (W138X), and 24 had 21 other mutations. In 20 patients (19%), no mutations were found. Of 82 alleles bearing the 65-kb deletion, 38 derived from Germany, 28 from the British Isles, and 4 from Iceland. Eighteen new mutations were identified, including the first reported missense mutations, two in-frame deletions, and mutations in patients of African American, Mexican, and Indian ancestry. CTNS mutations are spread throughout the leader sequence, transmembrane, and nontransmembrane regions. According to a cystinosis clinical severity score, homozygotes for the 65-kb deletion and for W138X have average disease, whereas mutations involving the first amino acids prior to transmembrane domains are associated with mild disease. By northern blot analysis, CTNS was not expressed in patients homozygous for the 65-kb deletion but was expressed in all 15 other patients tested. These data demonstrate the origins of CTNS mutations in America and provide a basis for possible molecular diagnosis in this population.     

Hermansky-Pudlak syndrome: models for intracellular vesicle formation

Hermansky-Pudlak syndrome (HPS) is an autosomal recessive disorder characterized by pigment dilution, nystagmus, decreased visual acuity, a bleeding diathesis, and lysosomal accumulation of ceroid lipofuscin. Electron microscopic evidence demonstrating lack of platelet-dense bodies provides the sine qua non for diagnosing HPS. Ceroid lipofuscinosis is considered to cause several serious complications, including progressive pulmonary fibrosis leading to death in the fourth or fifth decades. Currently, only symptomatic treatment can be offered. Although rare in the general population, HPS occurs in northwest Puerto Rico with a prevalence of 1 in 1800. HPS1, the first gene found to be responsible for HPS, was mapped to chromosome 10q23 and subsequently isolated and sequenced. It consists of 20 exons encoding a 700-amino acid, 79.3-kDa peptide with no homology to any known protein. All 10 HPS1 mutations reported to date, including the 16-bp duplication found in all northwest Puerto Rican patients, result in truncated proteins. The two mutations in the mouse pale ear gene (ep), which is the murine homology of HPS1, cause similarly truncated proteins. The pathologic nature of these truncation mutations may result from unstable mRNA. However, in combination with the absence of any disease-causing missense mutations, it may indicate that the C-terminus of the HPS1 peptide is functionally important. The disorder HPS displays locus heterogeneity, consistent with the existence of 14 mouse strains manifesting both hypopigmentation and a platelet storage pool deficiency. Two mouse models, pearl and mocha, have mutations in the beta3A and delta subunits of the adaptor-3 complex, respectively. This suggests that defective vesicular trafficking, specifically cargo packaging, vesicle formation, vesicle docking, or membrane fusion, may comprise the basic defect in HPS. Studies of the proteins involved in intercompartmental transport for melanosomes, platelet-dense bodies, and lysosomes should lead to a better understanding of the mechanisms of organellogenesis and to more effective therapies for HPS.     

What's new in pediatric nephrology?]

Advances in pediatric nephrology has been mainly characterized during the last years by a burst of knowledge in the area of genetic renal diseases: 1/almost complete understanding of Alport syndrome related to mutations of COL4A5 or COL4A3/A4 genes of collagene; 2/the mapping and cloning of the nephronophthisis gene which is deleted in 75% of cases; 3/the mapping and cloning of the cystinosis gene coding for a protein of the lysosomal membrane; 4/the mapping and cloning of the Finnish-type congenital nephrotic syndrome gene; 5/the linkage to the SNR 1 gene on chromosome 1 of a large number of familial corticoresistant nephrotic syndromes, and the disclosure of mutations of the WT1 gene in diffuse mesangial sclerosis and in Frazier syndrome. The understanding of Bartter syndrome has been also enlightened by the discovery of mutations in several ionic channels located in the distal tubule. It has been also shown that a corticoresistant nephrotic syndrome or a chronic tubular interstitial nephropathy are possible phenotypes for mitochondrial cytopathies. In the area of therapeutics, recombinant growth hormone was shown to improve statural growth of children with chronic renal failure; in addition, renal transplantation benefits from new immunosuppressants as tacrolimus and mycophenolate mofetil.     

Human hepatocyte growth factor (HGF) in the aqueous humor

Previous studies have demonstrated that individuals with cystinosis, an inherited metabolic disorder, have difficulty processing visual information, and may be selectively impaired in the ability to mentally rotate figures, despite having normal IQs and normal primary sensory function. In our novel task-the 'Black Box'-subjects identified objects solely by feeling the contours. Twenty-three subjects with cystinosis, aged 4 to 34 years, were individually matched with controls on age, sex, handedness, and test form. Subjects with cystinosis performed significantly worse in identifying objects than did controls. In addition, when only subjects over 7 years of age were included, those with cystinosis took significantly longer to correctly identify objects than did controls. Our findings suggest that individuals with cystinosis have difficulty with tactile recognition of common objects. These results support the hypothesis that a genetic disorder may have specific behavioral correlates.     

A selective role of brainstem noradrenergic neurons in oxytocin release from the neurohypophysis following noxious stimuli in the rat

Infantile nephropathic cystinosis is a genetic metabolic disorder in which the amino acid cystine accumulates in various organs, including the kidney, cornea, thyroid, and brain. Despite normal intellect, individuals with cystinosis may have specific impairments in the processing of visual information. To examine further the specific types of deficits in visual processing found in individuals with cystinosis, we administered the Development Test of Visual-motor Integration to 26 children with cystinosis (4 to 16 yr. old) and 26 matched controls. The cystinosis group achieved a significantly lower standard score, raw score, and mean ceiling than did the control group. Qualitative analyses showed that in the cystinosis group, size within errors and rotation errors were more prevalent than in the control group. Correlational analyses showed that with advancing age, the cystinosis subjects tended to fall further behind their chronological age. Our data, together with the findings of previous studies, suggest that the visuospatial difficulties in children with cystinosis may be due to inadequate perception or processing of visually presented information. Furthermore, the increasing discrepancy with age may reflect a progressive cognitive impairment, possibly as a result of cystine accumulation in the brain over time.     

Connexin mutations in X-linked Charcot-Marie-Tooth disease

X-linked Charcot-Marie-Tooth disease (CMTX) is a form of hereditary neuropathy with demyelination. Recently, this disorder was mapped to chromosome Xq13.1. The gene for the gap junction protein connexin32 is located in the same chromosomal segment, which led to its consideration as a candidate gene for CMTX. With the use of Northern (RNA) blot and immunohistochemistry technique, it was found that connexin32 is normally expressed in myelinated peripheral nerve. Direct sequencing of the connexin32 gene showed seven different mutations in affected persons from eight CMTX families. These findings, a demonstration of inherited defects in a gap junction protein, suggest that connexin32 plays an important role in peripheral nerve.     

Isolation, mapping, and functional expression of the mouse X chromosome glycerol kinase gene

Glycerol kinase (Gyk) participates in the metabolism of endogenously derived and dietary glycerol. Deficiency of the human enzyme activity is an X-linked recessive disorder with a clinical picture varying from childhood metabolic crisis to asymptomatic adults incidentally identified by hyperlipidemia screening (pseudohypertriglyceridemia). Gyk is a member of a small group of kinases termed ambiquitous enzymes that are found in the cytosol or as membrane-bound enzymes associated with the voltage-dependent anion channel of the mitochondrial outer membrane. It was recently reported that in humans there are X-linked and autosomal copies of Gyk sequences, both apparently functional genes and processed pseudogenes. To understand the role of Gyk in normal metabolism and the variable clinical features seen with Gyk deficiency, we have characterized the mouse Gyk gene. We present the sequence of a full-length mouse Gyk cDNA that is alternatively spliced in brain. The Gyk gene was mapped to the mouse X chromosome by both fluorescence in situ hybridization and an interspecies backcross panel, demonstrating conservation of synteny with dmd. To confirm the functional identity of the cDNA, transient transfection of the cDNA into COS7 cells was shown to cause a marked elevation in glycerol kinase activity.     

Slowly deteriorating insulin secretion and C-peptide production characterizes diabetes mellitus in infantile cystinosis

Infantile cystinosis, a rare lysosomal storage disease of cystine, leads to Fanconi syndrome and end-stage renal failure. After renal transplantation, no recurrence of the disease occurs in the graft, but other organ involvement becomes evident later in life. Diabetes mellitus has been associated with cystinosis, but the mechanisms of impaired glucose tolerance have not yet been characterized. Here, we studied glucose tolerance, glucose constant decay (k-values), insulin and C-peptide by intravenous glucose tolerance test (IVGTT) in eight patients with infantile cystinosis (three with impaired GFR (CRF) and five after kidney transplantation (KTX)). For comparison, 15 age-matched children with CRF and 15 age-matched KTX patients were analysed. Both early and second insulin secretion phases were diminished in patients with infantile cystinosis, whereas in CRF, k-values were no different from control patients. After renal transplantation, k-values were significantly lower in cystinotic patients with a markedly reduced early insulin secretion phase. There was a significant negative correlation between k-values and age in patients with cystinosis. Repetitive IVGTTs in these patients demonstrated progressive but rather slow loss of first phase insulin secretion and C-peptide production, suggesting a slowly reducing secretion potential of the beta cell due to cystine storage. CONCLUSION: Unlike type I diabetes mellitus, glucose intolerance in patients with infantile cystinosis is characterized by a slow, progressive loss of insulin secretion and C-peptide production. For these patients, the data indicate a 50% risk of developing glucose intolerance by the age of 18 years. We recommend to perform intravenous glucose tolerance tests at 5-year intervals.     

Actin-binding protein (ABP-280) filamin gene (FLN) maps telomeric to the color vision locus (R/GCP) and centromeric to G6PD in Xq28

Actin-binding protein-280 (ABP-280) is a dimeric actin filament crosslinking protein that promotes orthogonal branching of actin filaments and links actin filaments to membrane glycoproteins. We have mapped the ABP-280 filamin gene (FLN) to Xq28 by Southern blot analysis of somatic cell hybrid lines, by fluorescence in situ hybridization, and through identification of portions of the FLN gene within cosmids and YACs mapped to Xq28. The FLN gene is found within a 200-kb region centromeric to the G6PD locus and telomeric to DSX52 and the color vision locus.     

Pantetheinase activity and cysteamine content in cystinotic and normal fibroblasts and leukocytes

Cysteamine is the most effective agent known for the reduction of the elevated cystine content of cells from patients with cystinosis. A defect in endogenous cysteamine generation could account for many of the metabolic features of this disorder. To test this hypothesis, we have developed improved methods for measuring pantetheinase (cysteamine-generating) activity and intracellular cysteamine levels and used these methods to measure such parameters in cystinotic and normal leukocytes and cultured skin fibroblasts. Pantetheinase activity as defined in the test was similar in extracts of cystinotic and normal cells [leucocytes, normal, 78 +/- 15 (S.E.), cystinotic, 56+/- 6.4; fibroblasts, normal, 9.4 +/- 1.5; cystinotic, 7.7 +/- 1.7]. Cysteamine levels were normal in leukocytes from cystinotics receiving no cysteamine or doses of oral cysteamine too low to reduce leukocyte cystine content. The results indicate that the cause of cystinosis is unlikely to be related to a failure to generate of sustain normal intracellular cysteamine levels.     

Reduced cholesterol accumulation and improved deficient peroxisomal functions in a murine model of Niemann-Pick type C disease upon treatment with peroxisomal proliferators

Niemann-Pick type C disease is an inherited disorder characterized by lysosomal accumulation of cholesterol and the mutant gene has recently been identified. The predicted gene product is a transmembrane protein showing homology to proteins involved in the regulation of cholesterol homeostasis, such as 3-hydroxy-3-methylglutaryl-coenzyme A and the sterol regulatory element binding protein cleavage-activating protein. Recent investigations have established a peroxisomal deficiency, which raised the question of whether peroxisomal proliferation could affect this cholesterol-processing error. Mutant mice with Niemann-Pick type C disease were treated with the peroxisomal inducer perfluorooctanoic acid, which increased peroxisomal beta-oxidation and catalase activity to the same level as in control mice. Not only the peroxisomal, but also the lysosomal malfunctions were corrected and the cholesterol content was decreased. Clofibrate, another peroxisomal inducer, restored both peroxisomal enzyme activities and ubiquinone content. It appears that in Niemann-Pick type C disease treatment with appropriate peroxisomal inducers restores basic cellular functions, indicating a relationship between peroxisomes and cholesterol homeostasis, and thereby may effectively interfere with the development of the disease.     

Molecular cloning and expression of palmitoyl-protein thioesterase 2 (PPT2), a homolog of lysosomal palmitoyl-protein thioesterase with a distinct substrate specificity

Palmitoyl-protein thioesterase is a lysosomal hydrolase that removes long chain fatty acyl groups from modified cysteine residues in proteins. Mutations in this enzyme were recently shown to underlie the hereditary neurodegenerative disorder, infantile neuronal ceroid lipofuscinosis, and lipid thioesters derived from acylated proteins were found to accumulate in lymphoblasts from individuals with the disorder. In the current study, we describe the cloning and expression of a second lysosomal thioesterase, palmitoyl-protein thioesterase 2 (PPT2), that shares an 18% identity with palmitoyl-protein thioesterase. Transient expression of a PPT2 cDNA led to the production of a glycosylated lysosomal protein with palmitoyl-CoA hydrolase activity comparable with palmitoyl-protein thioesterase. However, PPT2 did not remove palmitate groups from palmitoylated proteins that are substrates for palmitoyl-protein thioesterase. In cross-correction experiments, PPT2 did not abolish the accumulation of protein-derived lipid thioesters in palmitoyl-protein thioesterase-deficient cell lines. These results indicate that PPT2 is a lysosomal thioesterase that possesses a substrate specificity that is distinct from that of palmitoyl-protein thioesterase.     

Molecular cloning, cDNA sequence, and chromosomal assignment of the human radixin gene and two dispersed pseudogenes

Radixin is a cytoskeletal protein that may be important in linking actin to the plasma membrane. Recent cloning of the murine and porcine radixin cDNAs revealed a protein highly homologous to ezrin and moesin. We have cloned and sequenced the human radixin cDNA and found the predicted amino acid sequence for the human protein to be nearly identical to those predicted for radixin in the two other species. By Southern analyses of Chinese hamster x human somatic cell hybrid DNA and of PCR products derived from hybrids, the coding gene (RDX) was mapped to 11q. Fluorescence chromosomal in situ hybridization with a cDNA plasmid further localized this gene to band 11q23. However, PCR amplification with "radixin-specific" primers on the hybrid DNA panel yielded an additional, very similar DNA sequence that was further characterized by direct sequencing of PCR products. This sequence represents a truncated version and the respective locus (RDXP2) was assigned to Xp21.3. Furthermore, by employing a different set of primers, a third sequence was found that was 90% identical to the radixin sequence but contained termination codons and seemed to lack introns. This pseudogene (RDXP1) was mapped to 11p by Southern and PCR analyses.     

Agonist-induced phosphorylation of the angiotensin AT1a receptor is localized to a serine/threonine-rich region of its cytoplasmic tail

Autosomal recessive juvenile parkinsonism (AR-JP) is a distinct clinical and genetic entity characterized by selective degeneration of nigral dopaminergic neurons and young-onset parkinsonism with remarkable response to levodopa. Recently, we mapped the gene locus for AR-JP to chromosome 6q25.2-q27 by linkage analysis and we identified a novel large gene, Parkin, consisting of 12 exons from this region; mutations of this gene were found to be the cause of AR-JP in two families. Now we report results of extensive molecular analysis on 34 affected individuals from 18 unrelated families with AR-JP. We found four different homozygous intragenic deletional mutations, involving exons 3 to 4, exon 3, exon 4, and exon 5 in 10 families (17 affected individuals). In addition to the exonic deletions, we identified a novel one-base deletion involving exon 5 in two families (2 affected individuals). All mutations so far found were deletional types in which large exonic deletion accounted for 50% (17 of 34) and the one-base deletion accounted for 6% (2/34); in the remaining, no homozygous mutations were found in the coding regions. Our findings indicate that loss of function of the Parkin protein results in the clinical phenotype of AR-JP and that subregions between introns 2 and 5 of the Parkin gene are mutational hot spots.     

The ''drug value'' in the European pharmaceutical system

Distal myopathy with rimmed vacuoles is an autosomal recessively inherited disorder with preferential involvement of the anterior tibial muscle. Recently the gene was discovered to be mapped to chromosome 9, the same region as in familial inclusion body myopathy (rimmed vacuole myopathy sparing the quadriceps). The onset of the disease was in young adults 20-40 years of age, averaging 26 years. The disease was progressive and most of the patients became non-ambulant within 12 years after the onset. The striking and common pathologic finding was the presence of rimmed vacuoles in muscle fibers with little evidence of necrotic or regenerative processes. Nuclear change with tubulofilamentous inclusions probably induces focal myofibrillar degeneration which activates the lysosomal system, resulting in active autophagocytosis and myelin body formation, i.e. rimmed vacuole formation.     

Notch3 mutations in CADASIL, a hereditary adult-onset condition causing stroke and dementia

Stroke is the third leading cause of death, and vascular dementia the second cause of dementia after Alzheimer's disease. CADASIL (for cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy) causes a type of stroke and dementia whose key features include recurrent subcortical ischaemic events and vascular dementia and which is associated with diffuse white-matter abnormalities on neuroimaging. Pathological examination reveals multiple small, deep cerebral infarcts, a leukoencephalopathy, and a non-atherosclerotic, non-amyloid angiopathy involving mainly the small cerebral arteries. Severe alterations of vascular smooth-muscle cells are evident on ultrastructural analysis. We have previously mapped the mutant gene to chromosome 19. Here we report the characterization of the human Notch3 gene which we mapped to the CADASIL critical region. We have identified mutations in CADASIL patients that cause serious disruption of this gene, indicating that Notch3 could be the defective protein in CADASIL patients.     

Localization of frontotemporal dementia with parkinsonism in an Australian kindred to chromosome 17q21-22

An Australian family with autosomal dominant presenile nonspecific dementia was recently described. The disease results in behavioral changes, usually disinhibition, followed by the onset of dementia accompanied occasionally by parkinsonism. Twenty-eight affected individuals were identified with an age of onset of 39 to 66 years (mean, 53 +/- 8.9 years). We mapped the disease locus to an approximately 26-cM region of chromosome 17q21-22 with a maximum two-point LOD score of 2.87. Affected individuals share a common haplotype between markers D17S783 and D17S808. This region of chromosome 17 contains the loci for several neurodegenerative diseases that lack distinctive pathological features, suggesting that these dementias, collectively referred to as frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17), are caused by mutations in the same gene. The entire coding region of five genes, mapped to the FTDP-17 candidate region, were also sequenced. This analysis included the microtubule-associated protein tau that is the major component of the paired helical filaments observed in Alzheimer's disease. No pathogenic mutations were identified in either the tau gene or in any of the other genes analyzed.     

Association of mutations in a lysosomal protein with classical late-infantile neuronal ceroid lipofuscinosis

Classical late-infantile neuronal ceroid lipofuscinosis (LINCL) is a fatal neurodegenerative disease whose defective gene has remained elusive. A molecular basis for LINCL was determined with an approach applicable to other lysosomal storage diseases. When the mannose 6-phosphate modification of newly synthesized lysosomal enzymes was used as an affinity marker, a single protein was identified that is absent in LINCL. Sequence comparisons suggest that this protein is a pepstatin-insensitive lysosomal peptidase, and a corresponding enzymatic activity was deficient in LINCL autopsy specimens. Mutations in the gene encoding this protein were identified in LINCL patients but not in normal controls.