Identification of HE1 as the second gene of Niemann-Pick C disease.

Niemann-Pick type C2 disease (NP-C2) is a fatal hereditary disorder of unknown etiology characterized by defective egress of cholesterol from lysosomes. Here we show that the disease is caused by a deficiency in HE1, a ubiquitously expressed lysosomal protein identified previously as a cholesterol-binding protein. HE1 was undetectable in fibroblasts from NP-C2 patients but present in fibroblasts from unaffected controls and NP-C1 patients. Mutations in the HE1 gene, which maps to chromosome 14q24.3, were found in NP-C2 patients but not in controls. Treatment of NP-C2 fibroblasts with exogenous recombinant HE1 protein ameliorated lysosomal accumulation of low density lipoprotein-derived cholesterol.

The natural history of Niemann-Pick disease type C in the UK.

Niemann-Pick disease type C (NPC) is an autosomal recessive, neurovisceral lipid storage disorder. Mutations in two genes (NPC1 and NPC2) produce indistinguishable clinical phenotypes by biochemical mechanisms that have not yet been entirely clarified. The wide spectrum of clinical presentations of NPC includes hepatic and pulmonary disease as well as a range of neuropsychiatric disorders. Late-onset disease has been increasingly recognized as the biochemical diagnosis of NPC has been more widely applied in adult neurology clinics. The clinical presentation and follow-up of 94 patients with NPC is described, 58 of whom were still alive at the time this report was prepared. The age at diagnosis ranged from the prenatal period (with hydrops fetalis) up to 51 years. This review of NPC patients in the UK confirms the phenotypic variability of this inherited lipid storage disorder reported elsewhere. Although a non-neuronopathic variant has been described, most patients in this series who survived childhood inevitably suffered neurological and in some cases neuropsychiatric deterioration. While symptomatic treatment, such as anticholinergic and antiepileptic drugs, can alleviate some aspects of the disease, there is a clear need to develop a specific treatment for this progressively debilitating neurodegenerative disorder.

Abnormal lysosomal sorting with an enhanced secretion of cathepsin D precursor molecules bearing monoester phosphate groups.

It has been reported that besides defects in the phosphorylation such as in the I-cell disease, a failure in the uncovering of mannose 6-phosphate residues may result in an increase of lysosomal enzyme activities in serum [Alexander et al., Hum. Genet. 73, 53-59 (1986)]. We examined fibroblasts that were derived from the original biopsy, observed an enhanced secretion of lysosomal enzymes including cathepsin D, but found that both the phosphorylation and uncovering of mannose 6-phosphate residues were normal. The enhanced secretion of cathepsin D was characterized by an increase in the secretion of phosphorylated molecules that were sensitive to a treatment with alkaline phosphatase. The enhanced secretion of the phosphatase-sensitive form of procathepsin D was further increased in the presence of antibodies directed to cation-independent mannose 6-phosphate receptors. In contrast, antibodies specific to cation-dependent mannose 6-phosphate receptors selectively inhibited the secretion of the phosphatase-sensitive procathepsin D molecules. A chromatographic analysis of oligosaccharides from the secreted procathepsin D confirmed that the cells secrete proenzyme molecules rich in oligosaccharides with two uncovered phosphate residues. It is suggested that the enhanced secretion of procathepsin D in the variant fibroblasts results from an abnormal sorting rather than processing of phosphorylated lysosomal enzymes.

Transplantation of fetal fibroblasts and correction of enzymatic deficiencies in patients with Hunter's or Hurler's disorders.

An attempt was made at correcting the specific lysosomal enzyme deficiencies in 7 children with Hunter's or Hurler's diseases by transplantation of fetal fibroblasts. In spite of pretreating the young patients with stored blood, following a procedure employed successfully to avoid rejection of kidneys from incompatible donors, the use of serum-free media for culturing the cells before being harvested and incubation of the cells with chorionic gonadotrophin, the transplantation of fetal fibroblasts was not associated with biochemical or clinical changes. None of the seven patients showed immune reactions against the transplanted cells, HLA antigens, or the missing enzymes.

Application of a flowchart for the detection of lysosomal storage diseases in 105 high-risk Brazilian patients.

Lysosomal storage diseases (LSD) are a group of more than 40 disorders, many of them with overlapping phenotype, in which clinical diagnosis is often difficult. Definitive diagnosis is based on enzyme assays, a large number of such assays usually being necessary during the investigation of each patient. In addition, there will frequently be a need for tissue culture in order to provide enough material for analysis. Taking into account these difficulties, we designed a flowchart for the detection of LSD that is based on 2 sets of tests requiring only random urine and heparinized blood. Here we describe this routine and report the results of its application to 105 Brazilian patients in whom a LSD was suspected. We think that the application of this rationale represents a saving of work and costs, and should be of special interest to genetic centers in developing countries.

Prenatal diagnosis of the urea cycle diseases: a survey of the European cases.

A European survey of prenatal diagnosis cases involving urea cycle diseases was performed. Citrullinemia was the most frequently investigated disease (108 cases). Other diseases are, in order of frequency, argininosuccinic aciduria (75 cases), ornithine transcarbamylase defect (52 cases), carbamoylphosphate synthetase defect (8 cases), triple H (3 cases), and arginase deficiency (1 case). Only one disease (ornithine transcarbamylase defect) is presently diagnosed using molecular biology methods.

Peroxisomal disorders: clinical and biochemical studies in 15 children and prenatal diagnosis in 7 families.

We describe the main clinical and biochemical findings in 15 patients with peroxisomal disorders, together with the results of 11 prenatal investigations for Zellweger syndrome. The initial laboratory diagnosis depended in most cases on demonstration of elevated very long chain fatty acids in plasma, but follow-up studies using cultured fibroblasts were essential for complete classification. The patient group comprises nine cases of Zellweger syndrome, one of neonatal adrenoleucodystrophy, two of infantile Refsum disease, one of bifunctional protein deficiency, and two of rhizomelic chondrodysplasia punctata. The study illustrates the clinical and biochemical variability of this group of patients and the detailed studies that are required for classification.

Arylsulphatase A pseudodeficiency in vascular dementia and Alzheimer's disease.

The carrier rates of a genetic marker for arylsulphatase A pseudodeficiency (ASA-PD) were determined in three series of patients with vascular dementia (VaD) or Alzheimer's disease (AD). In the first community-based sample, the 1524 + 95A-->G mutation, which is known to be associated with ASA-PD, was present in 35% of VaD cases and none of the AD cases. In a second sample of cases drawn from a Dementia Register, the mutation rates were 18% (VaD) and 16% (AD). Brain DNA from a post-mortem sample revealed the ASA-PD mutation in 60% of VaD cases and 34% of AD cases. These rates are higher than previous studies of culturally similar populations and suggest that ASA-PD may be a risk factor for dementia.

Neurodegenerative course in ceramidase deficiency (Farber disease) correlates with the residual lysosomal ceramide turnover in cultured living patient cells.

Farber's lipogranulomatosis is an inborn lipid storage disease characterized by tissue accumulation of ceramide due to deficient activity of lysosomal ceramidase. Symptoms include painful swelling of joints, subcutaneous nodules, a hoarse cry, hepatosplenomegaly and nervous system dysfunction of markedly variable degree. In most cases the neural dysfunction rather than the general dystrophy, seems to limit the duration of Farber disease. We examined whether the severity can be shown as a function of ceramide turnover by lysosomal ceramidase. The lysosomal degradation of sphingomyelin-derived ceramide was studied in situ in patient skin fibroblasts and lymphoid cells loaded with LDL-associated radioactive sphingomyelin. We could show for the first time a significant correlation between the ceramide accumulated in situ and the severity of Farber disease. Our method provides an alternative means for determining ceramide degradation by lysosomal ceramidase, but in intact cells. The relatively simple method is at least of the same diagnostic use for Farber disease as the in vitro assay of acid ceramidase using cell homogenates and may also have some prognostic use.

Assay of galactose-1-phosphate uridyl transferase in cultured amniotic cells for prenatal diagnosis of galactosaemia.

We report studies designed to establish optimal conditions for the assay of amniotic cell galactose 1-phosphate uridyl transferase (Gal-PUT) for early prenatal diagnosis of galactosaemia. Methods based on linkage of the reaction to cause of non-specific reactions occurring even in the absence of Gal-1-P. In the final method, sonicates of confluent cultures are incubated with (14-C) Gal-1-P is degraded by treatment with alkaline phosphatase. Gal-PUT specific activities of both control and galactosaemic amniotic cells are higher in non-confluent that confluent cultures.

A rapid method for assay of branched-chain keto acid decarboxylation in cultured cells and its application to prenatal diagnosis of maple syrup urine disease.

A sensitive assay for measurement of branched-chain keto acid decarboxylation in small numbers of fibroblasts or amniotic cells grown in the wells of a microtitre plate using [1-14C]leucine as substrate is described. The method was applied to the amniotic cells from a pregnancy at risk for maple syrup urine disease and a heterozygous fetus predicted.

A simple method for screening for Farber disease on cultured skin fibroblasts.

Farber disease is an inborn lysosomal storage disorder characterized by accumulation of ceramide in the patient's tissues due to the deficient activity of acid ceramidase. Currently, confirmation of the diagnosis is performed in an extremely limited number of laboratories. We therefore developed a procedure which does not require any particular sphingolipid substrate and is based on the quantitation of ceramide levels in cultured skin fibroblasts. In the method we devised, the ceramide present in cellular lipid extracts subjected to mild alkaline hydrolysis was quantified using the commercially available diacylglycerol kinase kit. We show that both primary cultures of skin fibroblasts and SV40-transformed fibroblasts derived from a series of patients with Farber disease exhibit ceramide excess as compared to their normal counterparts (2345-17 153 pmol/mg cell protein in Farber cells vs. 432-1298 pmol/mg cell protein in controls). Use of this simple method should greatly facilitate the biochemical diagnosis of Farber disease.

Corneal clouding in GM1-generalized gangliosidosis.

Corneal clouding is added to the list of clinical and chemical abnormalities which occur both in GM1-generalized gangliosidosis and in Hurler's syndrome (and some other mucopolysaccharidoses). The parents of our patient were first cousin Yemeni and had partial beta-galactosidase deficiency in their leucocytes and cultured fibroblasts.

Biallelic discrimination assays for the three common Ashkenazi Jewish mutations and a common non-Jewish mutation, in Tay-Sachs disease, using fluorogenic TaqMan probes.

We have developed rapid semiautomated fluorogenic TaqMan assays for the three common Jewish mutations that occur in Tay-Sachs disease, the TATC 4-bp insertion in exon 11 (1,278insTATC), the IVS 12 + 1G --> C, splice site mutation in intron 12 (1421 + 1 G --> C), and the G --> A change at the 3' end of exon 7 (G269S), as well as for a non-Jewish mutation, IVS9 + I G --> A, believed to be prevalent in patients of Celtic descent. The TaqMan assays are designed to run on the ABI SDS 7700 sequence detection system, using allele-specific probes that carry a reporter dye at the 5' end and a quencher dye at the 3' end. Using a 96-well format, all four assays can be performed simultaneously on the same plate, with real-time fluorescence detection or just an end-point plate read. DNA samples from 78 patients identified as carriers by biochemical screening and genotyped by conventional techniques were used to assess the accuracy and efficiency of the probes in allelic discrimination assays. There were no discrepancies noted between previously assigned genotypes and the results obtained by application of this methodology.

Farber lipogranulomatosis type 1--late presentation and early death in a Croatian boy with a novel homozygous ASAH1 mutation.

BACKGROUND: We report a boy with an unusually late presentation of Farber lipogranulomatosis type l. CASE STUDY: The first symptoms appeared at the end of the first year of life in the form of joint swelling; other symptoms such as cherry-red spot, hoarseness, subcutaneous nodules appeared much later. The history of the disease, from the first symptoms till his early death, lasted 26.5 months. The neuronal dysfunction accompanied by the rapid neurological deterioration with seizures and myoclonias, rather than the general dystrophy, seemed to limit the duration of disease in our patient and provoked his early death. Diagnosis was confirmed by analysis of ceramide metabolism in cultured fibroblasts and of the ASAH1 gene, which indicated homozygosity for a novel point mutation. CONCLUSION: The deficient activity of acid ceramidase correlated well with poor prognosis of the disease in our boy, in contrast to late appearance of dermal nodules and the subsequent severe clinical course with fatal outcome. Farber lipogranulomatosis should be suspected in children with joint swelling as the first and only symptom of disease. In order to advance our knowledge towards establishing genotype-phenotype correlations in Farber's disease, detailed analysis of the ASAH1 gene is needed.