Frequency of Fabry disease in patients with small-fibre neuropathy of unknown aetiology: a pilot study.

BACKGROUND: Early occurrence of small-fibre neuropathy (SFN) is a common feature of Fabry disease (FD) - an X-linked storage disorder caused by reduced activity of the α-galactosidase A (α-GAL). Although SFN may result from different disorders, the cause is often unclear. Therefore, we investigated the frequency of FD in patients with SFN of unknown aetiology. METHODS: Patients with idiopathic SFN, established by sensory quantitative testing and/or skin biopsy, were examined for mutations in the α-GAL gene. Where mutations in the α-GAL gene were identified, levels of globotriaosylceramide (Gb(3)) were measured in urine and blood and the α-GAL activity was evaluated. When new mutations were detected, a diagnostic work-up was performed as well as a Gb(3) accumulation in the skin, lyso-Gb(3) in blood and Gb(3)_24 in urine were proved. RESULTS: Twenty-four of 29 eligible patients were enrolled in the study. Mutations in the α-GAL gene were observed in five patients. A typical mutation for FD (c.424T>C, [C142R]) was detected in one patient. In four patients, a complex intronic haplotype within the α-GAL gene (IVS0-10C>T [rs2071225], IVS4-16A>G [rs2071397], IVS6-22C>T [rs2071228]) was identified. The relevance of this haplotype in the pathogenesis of FD remains unclear until now. However, these patients showed increased concentrations of Gb(3) and/or lyso-Gb(3), while no further manifestations for FD could be proved. CONCLUSIONS: Fabry disease should be considered in patients with SFN of unknown aetiology, and screening for FD should be included in the diagnostic guidelines for SFN. The significance of the intronic haplotype regarding SFN needs further evaluation.

Phenotype determining alleles in GM1 gangliosidosis patients bearing novel GLB1 mutations.

GM1 gangliosidosis manifests with progressive psychomotor deterioration and dysostosis of infantile, juvenile, or adult onset, caused by alterations in the structural gene coding for lysosomal acid beta-galactosidase (GLB1). In addition, allelic variants of this gene can result in Morquio B disease (MBD), a phenotype with dysostosis multiplex and entire lack of neurologic involvement. More than 100 sequence alterations in the GLB1 gene have been identified so far, but only few could be proven to be predictive for one of the GM1 gangliosidosis subtypes or MBD. We performed genotype analyses in 16 GM1 gangliosidosis patients of all phenotypes and detected 28 different genetic lesions. Among these, p.I55FfsX16, p.W65X, p.F107L, p.H112P, p.C127Y, p.W161X, p.I181K, p.C230R, p.W273X, p.R299VfsX5, p.A301V, p.F357L, p.K359KfsX23, p.L389P, p.D448V, p.D448GfsX8, and the intronic mutation IVS6-8A>G have not been published so far. Due to their occurrence in homozygous patients, four mutations could be correlated to a distinct GM1 gangliosidosis phenotype. Furthermore, the missense mutations from heteroallelic patients and three artificial nonsense mutations were characterized by overexpression in COS-1 cells, and the subcellular localization of the mutant proteins in fibroblasts was assessed. The phenotype specificity of 10 alleles can be proposed on the basis of our results and previous data.

Photosensitized killing of cultured fibroblasts from patients with peroxisomal disorders due to pyrene fatty acid-mediated ultraviolet damage.

The influence of pyrene-fatty acids on the resistance of cells to ultraviolet (UV) radiation was investigated in cultured fibroblasts from patients with five types of peroxisomal disorders. All showed reduced survival compared to control. The effect varied with the biochemical defect involved and the chain length of the pyrene fatty acid. Reduced survival was observed in cells deficient in plasmalogens (rhizomelic chondrodysplasia punctata) and in cells deficient in peroxisomal fatty acid oxidation (bifunctional enzyme deficiency), which accumulated pyrene-fatty acids. X-linked adrenoleukodystrophy fibroblasts accumulated pyrene-fatty acids and showed increased UV sensitivity only when exposed to longer-chain pyrene fatty acids. UV radiation resistance was lowest in cells with combined impairment of plasmalogen synthesis and fatty acid oxidation (Zellweger syndrome, neonatal adrenoleukodystrophy), suggesting that UV sensitivity correlates inversely with the ratio of plasmalogens to radical producing substances. Fibroblasts deficient in plasmalogens gained normal UV resistance when their plasmalogen levels were normalized by hexadecylglycerol. UV resistance increased when Zellweger cells were fused with X-linked adrenoleukodystrophy cells, and also when Zellweger cells belonging to different complementation groups were fused. The results provide leads to the pathogenesis of the multiple malformations associated with peroxisomal disorders and a method for the selection of cells in which the metabolic defect has been corrected.

Copper concentration of liver tissue under long-term copper-histidine therapy in a patient with Menkes disease.

Copper-histidine is the treatment of choice in Menkes disease but bears the potential risk of copper overload and induced liver cirrhosis. We report normal copper concentrations of liver tissue over an 8-year treatment period with copper-histidine.

Aggregation properties of beta-galactosidase of human urine and degradation of its natural substrates by a purified preparation of the enzyme.

Acid beta-D-galactosidase (beta-D-galactoside galactohydrolase, EC 3.2.1.23) was purified to near homogeneity from normal human urine by two affinity chromatography steps. On polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate the major protein band had an apparent molecular weight of 59000, thus being 5000 daltons smaller than the protein purified from human liver. Upon gel filtration on Sephadex G-150 column the purified enzyme had an apparent molecular weight of 70000 of pH 7.0. At pH 4.0 partial aggregation to a dimer of an apparent molecular weight of 150000 was found. Addition of 0.1 M galactose caused at pH 3.5, but not at pH 4.0 and 7.0, an increased formation of multimeric beta-galactosidase which eluted with the void volume of the column. Crude beta-galactosidase from human urine showed a higher aggregation tendency than the purified enzyme. None of the conditions produced an enzyme species of an apparent molecular weight of less than 40000. pH-activity profiles were measured against p-nitrophenyl-beta-D-galactoside, 3H-labelled GM1-ganglioside, [3H]keratan sulfate and the pentasaccharide O-beta-(1 leads to 4)-[6-3H]galactopyranosyl-O-beta-(1 leads to 2)-2-deoxy-2-acetamidoglycopyranosyl-O-alpha-(1 leads to 6)-mannopyranosyl-O-beta-(1 leads to 4)-mannopyranosyl-2-deoxy-2-acetamidoglucopyranoside. While p-nitrophenyl-beta-D-galactopyranoside and GM1-ganglioside were optimally hydrolyzed at pH 4.0, keratan sulfate and the pentasaccharide were optimally degraded at pH 4.3 and pH 5.0, respectively. With the chromogenic substrate and with GM1-ganglioside Km values of 0.33 mM were calculated. At pH 3.5 the hydrolysis of the synthetic substrate did not follow Michaelis-Menten kinetics. Two enzyme species appeared with Km values of 0.006 mM and 3.2 mM, respectively. The affinity of beta-galactosidase for [3H]keratan sulfate and the 3H-labelled pentasaccharide was at least one order of magnitude lower than for the amphiphilic substrates. Keratan sulfate and GM1-ganglioside did not act as competitive inhibitors of p-nitrophenyl-beta-galactosidase at the concentration tested. These findings could be explained by the existence of different binding sites for the substrates used.

Uptake of fluorescent plasmalogen analogs by cultured human skin fibroblasts deficient in plasmalogen.

One of the consequences of hereditary peroxisomal dysfunction in the cerebro-hepato-renal (Zellweger) syndrome (CHRS) is a dramatic decrease in the biosynthesis and cellular content of ether lipids. In the present study effects of reduced cellular plasmalogen levels on membrane-membrane interactions were investigated. Cultured CHRS fibroblasts were incubated with unilamellar phospholipid vesicles consisting of 1-O-alkenyl-2-acyl- or 1,2-diacyl-sn-glycerophosphocholines and ethanolamines, carrying either the trans-parinaroyl or the 1,6-diphenyl-1,3,5-hexatriene propionyl group in position 2. Transfer of the fluorogenic phospholipids from vesicles to cells was followed by measuring the concomitant increase in fluorescence intensity. Transfer of phospholipids from cells to vesicles was monitored by incubating cells, prelabeled with [3H]oleic acid, in the presence of phospholipid vesicles. Fibroblasts from healthy donors or CHRS fibroblasts supplemented with the plasmalogen precursor 1-O-hexadecylglycerol served as controls. Plasmalogen-deficient cells exhibited a significantly increased tendency to take up exogenous choline or ethanolamine plasmalogens. Cellular plasmalogens were transferred from control cells to vesicles at a higher rate if the acceptor vesicles consisted of plasmalogens as compared to diacylglycerophosphocholine. Thus, it appears as if mechanisms existed which preserve cellular plasmalogen levels during interaction with exogenous phospholipid pools. Preliminary experimental evidence suggests that the observed exchange of phospholipids between cultured fibroblasts and vesicles occurs by a protein-catalyzed process.

Cyclooxygenase pathway in dermal fibroblasts from patients with metabolic disorders of peroxisomal origin.

Cyclooxygenase metabolism was studied in fibroblasts from patients with metabolic disorders of peroxisomal origin (adrenomyeloneuropathy, X-linked adrenoleukodystrophy, cerebrohepatorenal syndrome of Zellweger and rhizomelic chondrodysplasia punctata). In response to arachidonic acid (6.25-100 microM) or calcium ionophore A23187 (2.5-20 microM) prostaglandin E2 and 6-keto-prostaglandin F1 alpha are the main cyclooxygenase metabolites formed. No formation of thromboxane B2 or 2,3-dinor-thromboxane B2 was found. Apparently due to the heterogeneous nature of peroxisomal disorders no uniform pattern of cyclooxygenase metabolism and eicosanoid concentrations in cell lines from patients with peroxisomal defects was found.

New phenotype of adult alpha-L-iduronidase deficiency (mucopolysaccharidosis I) masquerading as Friedreich's ataxia with cardiopathy.

Clinical, ultrastructural and biochemical studies are reported in a 42-year-old woman presenting with congenital pes cavus who, at the age of 23 years, developed slowly progressive distal amyotrophies, hypesthesia, bilateral hearing loss and severe cardiopathy leading to death. There were skeletal anomalies, mild reduction of motor NCVs, but no corneal opacity, retinitis pigmentosa, organomegaly or vacuolated lymphocytes. Autopsy disclosed severe thickening of fibrous tissues (endocardium, cerebrospinal dura) with accumulation of vacuolated cells containing glycosaminoglycans in numerous membrane-bound cytoplasmic vacuoles, and/or compound multilamellar or zebra-body-like structures. The CNS, in addition to enlarged perivascular lacunes in cerebral white matter with lipid-containing macrophages, showed neuronal lipid storage in thalamus, hypothalamus, hippocampus, brain stem nuclei, spinal motor neurons and Purkinje cell dendrites. Ultrastructurally, lamellated inclusions containing gangliosides were seen in mesenchymal cells, oligodendrocytes, pericytes and Schwann cells. Neurons contained abundant ceroid but no lamellated inclusions. Neurochemistry revealed decrease of alpha-L-iduronidase activity in brain tissue to 4% of normal, normal activities of other lysosomal enzymes, and normal lipid and ganglioside patterns. While the morphology and neurochemistry data are characteristic of mucopolysaccharidosis I, the phenotype of adult alpha-L-iduronidase deficiency mimicking Friedreich's disease has not been described so far.

[Strategies for the diagnosis of lysosomal storage diseases: symptoms, methods and samples]. / Strategien zur Diagnose lysosomaler Speichererkrankungen: Symptome, Methoden, Untersuchungsmaterial.

Lysosomal diseases are a group of about 30 genetic defects with a total incidence of 3 to 4 cases/10,000 newborns. Their clinical appearance is very heterogeneous and comprises infantile, as well as juvenile or adult forms. Our concept for their diagnosis has now been in use for eight years and entails the following strategy: The three main symptom groups coarse facial features, visceromegaly and/or psychomotor retardation should be examined for their typical expression and for the occurrence of specific "key symptoms". Thereafter, biochemical analysis of urine for oligosaccharides, mucopolysaccharides and in some cases, of sphingolipids or direct enzyme assays in serum, peripheral leucocytes or skin fibroblasts are performed. The selection of appropriate methods is usually the domain of the biochemist and greatly depends on the available samples and the quality of clinical information. The diagnostic value and the limitations of methods and samples are discussed in detail. Finally, evidence of defects in the expression of relevant gene products, such as enzymes, activator proteins or transport proteins can be obtained and used for genetic counselling and/or for prenatal diagnosis in chorionic villi or cultured amniotic fluid cells. Our results confirm the data on the high incidence of mucopolysaccharidoses I and III A. In addition, a comparatively high number of otherwise rare diseases, such as fucosidosis or sialic acid storage disease was found. Among the group of sphingolipidoses, special attention should be paid to juvenile or adult forms.

[Liver diseases in children and their relatives with homozygous and heterozygous alpha 1-antitrypsin deficiency]. / Lebererkrankungen bei Kindern und deren Verwandten mit homozygotem und heterozygotem Alpha-1-Antitrypsinmangel.

Over a 6-year period 26 infants and children with homozygous (2 Z and 6 ZZ) or heterozygous alpha 1-antitrypsin deficiency (12 MZ, 6 MS) were observed prospectively and their families investigated. 7 of 8 homozygous patients had neonatal hepatitis, whereby 3 of these showed maximum transferase activities during the 5th to 9th months of life. At the age of 7 years 2 of these patients were clinically normal, but only one patient had normal transferases. One patient had cirrhosis with portal hypertension at the age of 16 years 6 months; her nephew showed hypersplenism. Family studies revealed a further 5 relatives of phenotype Z, 16 of phenotype MZ, 3 of phenotype SZ and 1 of phenotype MS; 6 of these had slightly elevated serum transferase activities. 6 patients of phenotype MZ and 2 patients of phenotype MS had neonatal hepatitis but generally with a much better prognosis than in homozygous patients. The other heterozygous patients (6 MZ and 4 MS) had a variety of additional factors determining the disease and the prognosis. Family studies showed a further 7 family members of phenotype MZ, and 2 of phenotype MS; 2 of these had slightly elevated transferase activities, 3 parents had hereditary hyperbilirubinaemia.

Hunter syndrome (Muccopolysaccharridosis Type II) in Macedonia and Bulgaria.

BACKGROUND: Mucopolysaccharidosis II (MPS II) is caused by a deficiency of iduronate-2-sulfatase (IDS; EC 3.1.6.13). METHODS AND RESULTS: We describe 11 boys from Bulgaria and Macedonia detected in the period from 1998 to 2008. The mean age at diagnosis was 4.77+/-1.29 years. All children were severely retarded: IQ ranged from 34-80, and they all had coarse faces and hepatomegaly. In addition, splenomegaly was found in 81.81% patients, dysostosis in 45.45%, kyphosis in 27.27%, deafness in 18.08%, growth below the third percentile in 45.45%, growth below the parental target height in all patients, stiff joints in 56.56% and hypertrophic myocardiopathy in 18.18% children. Two patients died at the age of 11 and 35 years. Plasma iduronate-2-sulfatase was low in all probands and normal in parents and relatives. Two new mutations were discovered: p.K236N (c.708G>C) in a child with a moderately severe phenotype, and p.Q80K (c.238C>A) which resulted in a severe phenotype and early death at the age of 11 years. Heterozygote carriers of the pathogenic allele were 29 female relatives. The calculated incidence rate for MPS II in Macedonia (censuses 1994 and 2002, children under 14 years: 483,923 and 426,280) and Bulgaria (censuses 1992 and 2006, children under 14 years: 1 126, 598 and 1,077,020) are 0.36 and 0.46 respectively, while the calculated prevalence rate are 3.6 and 4.6 per 1,000,000 boys (aged 0-14 years). Correlating phenotype and genotype remains a complex endeavour. CONCLUSIONS: We report calculated incidence and prevalence rates in two South Eastern European countries, and 2 novel genetic alterations correlated with their phenotypes.

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.

Pyridoxine-dependent epilepsy: normal outcome in a patient with late diagnosis after prolonged status epilepticus causing cortical blindness.

We report on a male proband with pyridoxine-dependent epilepsy (PDE) and neonatal seizure onset. At the age of 31 months, a prolonged status epilepticus led to severe neurological regression with cortical blindness, loss of speech and muscular hypotonia with slow recovery over the following 3 months. At 33 months of age pyridoxine therapy was initiated with excellent response and the boy remained seizure-free on pyridoxine monotherapy, except for two occasions with seizure recurrence 10 days after accidental pyridoxine withdrawal. alpha-aminoadipic semialdehyde dehydrogenase (antiquitin) deficiency was indicated by elevated pipecolic acid concentrations in plasma and alpha-aminoadipic semialdehyde excretion in urine. Molecular analysis of the antiquitin gene revealed a novel missense mutation c.57insA, while the mutation of the other allele remained unidentified so far. Despite the delay in diagnosis and prolonged status epilepticus, neuropsychological evaluations at the ages of 11 and 18 years demonstrated full-scale IQ of 93 and 92, respectively, with better verbal IQ (103 and 101) than performance IQ (85 and 82).

Keratan sulphate levels in mucopolysaccharidoses and mucolipidoses.

The mucopolysaccharidoses (MPS) is characterized by accumulation of glycosaminoglycans (GAGs), and mucolipidosis (ML) by accumulation of GAGs and sphingolipids. Each type of MPS accumulates specific GAGs. The lysosomal enzymes N-acetylgalactosamine-6-sulphate sulphatase and beta-galactosidase involve the stepwise degradation of keratan sulphate (KS). Deficiency of these enzymes results in elevation of KS levels in the body fluids and in tissues, leading to MPS IV disease. In this study, we evaluated blood and urine KS levels in types of MPS and ML other than MPS IV. Eighty-five plasma samples came from MPS I (n = 18), MPS II (n = 28), MPS III (n = 20), MPS VI (n = 3), MPS VII (n = 5) and ML (n = 11) patients while 127 urine samples came from MPS I (n = 34), MPS II (n = 34), MPS III (n = 32), MPS VI (n = 7), MPS VII (n = 9) and ML (n = 11) patients. KS levels were determined using the ELISA method. Plasma KS levels varied with age in both control and patient populations. In all age groups, the mean values of plasma KS in MPS and ML patients were significantly higher than those in the age-matched controls. Plasma KS values in four newborn patients were above the mean + 2SD of the age-matched controls (mean, 41 ng/ml). Overall, 85.9% of individual values in non-type IV MPS and ML patients were above the mean + 2SD of the age-matched controls. For urine KS levels, 24.4% of individual values in patients were above the mean + 2SD of the age-matched controls. In conclusion, KS in blood is elevated in each type of non-type IV MPS examined, in contrast to the conventional understanding. This finding suggests that measurement of KS level provides a new diagnostic biomarker in a wide variety of mucopolysaccharidoses and mucolipidoses in addition to MPS IV.