Elevated sulfatide excretion in heterozygotes of metachromatic leukodystrophy: dependence on reduction of arylsulfatase A activity.

Sulfatide excretion in urine and arylsulfatase A (ASA) activity in leukocytes were determined in 10 homozygotes of metachromatic leukodystrophy (MLD), 7 obligate and 5 facultative MLD heterozygotes, 6 low ASA subjects (not related to MLD homozygotes), and in 9 controls. As compared to controls (sulfatides: 0-2 nmol/mg lipid; ASA: 101-287 nmol p-nitrocatechol/mg protein/hr), MLD homozygotes displayed highly increased sulfatide excretions (27-280 nmol) and low residual ASA activities (0-13 nmol). Of 12 MLD heterozygotes (ASA: 18-87 nmol) 10 showed increased sulfatides (3-24 nmol). All heterozygotes with ASA activity < 60 nmol (n = 8) had elevated sulfatide excretions (4-24 nmol). Thus, reduction of ASA activity below 40% of the mean value of controls seems to be the critical threshold for elevated sulfatide excretion in MLD heterozygotes. The low ASA subjects (ASA in the heterozygote range) excreted sulfatides in the control range, even those with ASA activities < 60 nmoles (n = 3; including a definite homozygote for ASA-pseudodeficiency; ASA:25 nmol). Statistical evaluation of sulfatide excretion and ASA activity in all subjects (n = 37) revealed a significant inverse relation (Spearman rank correlation; R = 0.8278, P < 0.001). The finding of elevated sulfatide excretion in certain MLD heterozygotes might point to increase of sulfatides also in the nervous system.

Occurrence, distribution, and phenotype of arylsulfatase A mutations in patients with metachromatic leukodystrophy.

Occurrence, distribution, and phenotype of arylsulfatase A (ASA) mutations were investigated in 27 patients with metachromatic leukodystrophy (MLD) from Central Europe, mainly from Austria (n = 15) and Poland (n = 9). Genomic DNA from leukocytes, fibroblasts, or paraffin-embedded, formalin-fixed brain or nerve tissue, respectively, was tested by natural or mutated primer-modulated PCR restriction, fragment length polymorphism for the eight most common European mutations: R84Q, S96F, 459+1G > A, I179S, A212V, 1204+1G > A, P426L, and 1401del11bp. The overall identification rate of unrelated MLD alleles was the highest, in adult (90%), medium in juvenile (50%), and lowest in late infantile (36%) MLD patients. The two common alleles, 459+1G > A and P426L, together accounted for 42% of all 50 unrelated MLD alleles investigated; I179S was observed in 6 of 50 MLD alleles (12%). Thus, I179S was far more frequent than hitherto thought and appears to be a third common mutation in Europe. Moreover, a different allelic distribution between Austrian and Polish juvenile patients was disclosed, indicating genetic heterogeneity of MLD even within Central Europe. The genotype-phenotype correlation suggested by Polten et al. [N Engl J Med 324:18-22, 1991] was not followed by all of our MLD patients. Moreover, some MLD patients with identical ASA mutations presented with different phenotypes. This may be due, at least in some cases, to the presence of an additional mutation on individual mutant alleles. Therefore, prediction of the clinical course from single mutation analysis is not possible.

Adrenoleukodystrophy in an adult female. A clinical, morphological, and neurochemical study.

A 43-year-old female with adrenoleukodystrophy (ALD) is described, who developed spastic tetraparesis, suffered grand mal seizures, and became stuporous and demented during the last 5 years of her life. Computed tomography revealed symmetrical hypodense lesions in the peritrigonal regions. Adrenal insufficiency was not evident except for skin pigmentation. The ultrastructure of a rectal biopsy specimen showed inclusions with lamellae and interspersed clefts in macrophages of the submucosal layer. At autopsy, the adrenals were found to contain large foam cells filled with similar inclusions. The brain cortex and the spinal cord were histologically normal. However, cerebral white matter exhibited widespread demyelination which spared only the arcuate fibres. In regions of less severe demyelination scattered inflammatory cells were seen. On electron microscopy, aggregates of typical paired leaflets with distinct intermediate lines were demonstrated in perivascular macrophages. Histochemical study showed these cells to contain free as well as esterified cholesterol. Gas chromatographic analysis of very long chain fatty acids (VLFA) from the demyelinated cerebral white matter showed a marked increase of C26:0 fatty acid in cholesterol esters and above-normal values for C24:0 and C24:1 in gangliosides. It is suggested that the condition was a heterozygote form of X-linked ALD. Patients with neurodegenerative symptoms with or without adrenal insufficiency can easily be screened for X-linked ALD by VLFA analysis in blood or cultured fibroblasts.

Elevated sulfatide excretion in compound heterozygotes of metachromatic leukodystrophy and ASA-pseudodeficiency allele.

OBJECTIVE: Use of sulfatide excretion in differentiating MLD/PD-heterozygotes from MLD-patients and PD/PD-homozygotes. DESIGN AND METHODS: Sulfatide was extracted from urine sediment with chlorotom/methanol (2:1, v/v). The quantity of sulfatide was measured densitometrically (lambda = 580 nm) after thin-layer chromatography. ASA and beta-galactosidase activities were assayed enzymatically. RESULTS: MLD/PD-heterozygotes excreted sulfatide in the range of 4.8-36.3 nmol/mg lipid (mean +/- SD = 17.8 +/- 10.7), whereas sulfatide in MLD-patients ranged from 74.3-411.6 nmol/mg lipid (mean +/- SD = 184.5 +/- 130.8) and in PD/PD-hormozygotes sulfatide excretion remained in normal range of 0.0-5.9 nmol/mg lipid (mean +/- SD = 1.64 +/- 2.12). ASA activities in these groups were very low or lowered. CONCLUSIONS: The quantitative measurement of sulfatide in urine allows differentiation between MLD/PD-heterozygotes and MLD-heterozygotes, as well as between MLD/PD-heterozygotes with very low ASA activity and MLD-patients or PD/PD-hormozygotes. The quantitative measurement of sulfatide in urine differs between MLD-carriers and controls.

Accumulation of very long chain fatty acids is common to 3 variants of adrenoleukodystrophy (ALD). "Classical" ALD, atypical ALD (female patient) and adrenomyeloneuropathy.

Fatty acids of cholesterol esters were analyzed by gas chromatography in affected CNS white matter of 3 variants of ALD ("classical" ALD, atypical ALD (adult female) and AMN) and of 10 controls with myelin breakdown of an etiology other than ALD. In all 3 ALD variants a marked accumulation of very long chain fatty acids (VLFA) as compared to control material was observed. This was due to the accumulation mainly of saturated C24-C32 fatty acids, particularly of C26:0, C25:0 and, to a lesser extent, C24:0 and C27:0 fatty acids. Our results demonstrate for the first time an accumulation of VLFA in an adult female patient (atypical ALD), who probably is an ALD heterozygote rather than a variant of AMN, and confirm and extend earlier findings in classical ALD and AMN, respectively. It appears that ALD may be a single nosological entity with clinically and morphologically different variants sharing specific ultrastructural (accumulation of paired leaflets) and neuro-biochemical (accumulation of VLFA) diagnostic markers.

Detection of GM2-gangliosidosis (Tay-Sachs and Sandhoff disease) gene carriers by serum hexosaminidase assay.

With a view to the biochemical detection of homo- and heterozygous carriers of GM2-gangliosidosis, serum hexosaminidase activities were investigated in patients from Tay-Sachs and from Sandhoff disease, respectively, in their relatives, and in normal controls. Two related methods for the differential determination of hexosaminidase A and B activities were tested. Homozygous carriers (patients) were detected by both methods in a similar manner. As regards the identification of heterozygous carriers more conclusive results were attained by the "heat inactivation method" (O'Brien, J.S., Okada, S., Chen, A. and Fillerup, D.L. (1970) New Engl. J. Med 283, 15).

Di-, mono- and nonphytanyl triglycerides in the serum: a sensitive parameter of the phytanic acid accumulation in Refsum's disease.

The relative proportions of the diphytanyl, monophytanyl and nonphytanyl triglycerides ("triglyceride pattern"), the phytanic acid content of the triglycerides and the phytanic acid levels in the serum of 3 patients with Refsum's disease (heredopathia atactica polyneuritiformis, phytanic acid storage disease) were estimated by thin-layer chromatography, densitometry and gas chromatography, respectively. The individual triglyceride patterns were clearly dependent on the phytanic acid content of the triglycerides: the more phytanic acid in the triglycerides, the higher the percentage of the diphytanyl and the lower the percentage of the nonphytanyl triglycerides. The monophytanyl triglycerides were also related to the phytanic acid content of the triglycerides, although in a complex manner. The triglyceride pattern can be taken as a parameter of the phytanic acid accumulation in Refsum's disease to be at least as sensitive as the serum phytanic acid level in the biochemical evaluation of the efficiency of the dietary treatment.

Detection of adrenoleukodystrophy by increased C26:0 fatty acid levels in leukocytes.

Very long chain fatty acids of peripheral blood leukocytes were analyzed by gas chromatography in nine members of a family including two hemizygotes and one obligate heterozygote for adrenoleukodystrophy (ALD), as well as in twelve controls. Comparative investigations were done in cultured fibroblasts. Elevated C26:0 levels were observed in leukocytes and fibroblasts of ALD hemizygotes. The obligate heterozygote displayed a clear-cut increase of C26:0 concentration in leukocytes but not in fibroblasts. Determination of C26:0 in leukocytes may serve as diagnostic tool in the detection of ALD gene carriers.

Very long chain fatty acids in genetic peroxisomal disease fibroblasts: differences between the cerebro-hepato-renal (Zellweger) syndrome and adrenoleukodystrophy variants.

Very long chain fatty acids were investigated by gas chromatography in fibroblasts of patients with genetic peroxisomal diseases (cerebro-hepato-renal (Zellweger) syndrome, childhood adrenoleukodystrophy, adrenomyeloneuropathy, neonatal adrenoleukodystrophy) and of controls. Concentrations of C 26:0 were increased to about the same extent in all disorders investigated. C 26:1 concentrations, on the other hand, were considerably elevated only in the cerebro-hepato-renal syndrome. In all control, adrenoleukodystrophy, and adrenomyeloneuropathy cases the C 22:0 concentration was higher than the respective C 26:0 concentration; the reverse was found in the cerebro-hepato-renal syndrome. These differences seem to reflect different impairment of peroxisomes in the cerebro-hepato-renal syndrome and adrenoleukodystrophy variants, respectively. Additional experiments to characterize C 26:1 by thin layer chromatography, gas chromatography and mass spectrometry revealed the presence of two straight-chain C 26:1 isomers with similar fragmentation patterns.

[Peroxisomal neurologic diseases and Refsum disease: very long chain fatty acids and phytanic acid as diagnostic markers]. / Peroxisomale neurologische Erkrankungen und M. Refsum: Uberlangkettige Fettsäuren und Phytansäure als diagnostische Marker.

Peroxisomal disorders are genetic metabolic diseases with generalized, multiple, or single functional disturbances of the peroxisome. According to the extent of the functional disturbances 3 groups of diseases can be differentiated: disorders with generalized loss of peroxisomal functions (Zellweger syndrome, ZS; neonatal adrenoleukodystrophy, NALD; infantile Refsum's disease), disorders with multiple enzymatic defects (e.g. rhizomelic chondrodysplasia punctata), and disorders with a single enzymatic defect in the peroxisome, the most important being adrenoleukodystrophy/adrenomyeloneuropathy (ALD/AMN). Adult Refsum's disease, a genetic neurological disorder with phytanic acid accumulation, is due to a mitochondrial enzyme deficiency, but is often considered together with peroxisomal diseases because of phytanic acid (PHYT) accumulation in most peroxisomal diseases. The main clinical and pathological criteria of the major disorders and the biochemical parameters of their differentiation are presented. Elevated levels of very long chain fatty acids (VLCFA) and/or PHYT are the primary diagnostic markers for all peroxisomal disorders and adult Refsum's disease, respectively. Our investigations disclosed 30 ALD/AMN hemizygotes, 16 ALD/AMN heterozygotes, 8 cases of ZS/NALD and 7 patients with adult Refsum's disease. In addition, 15 cases of peroxisomal disorders were confirmed by biochemical investigations in autopsy material. With regard to peroxisomal disorders, therapeutic concepts exist only for ALD/AMN: corticosteroid substitution for adrenal insufficiency, dietary treatment, and bone marrow transplantation (BMT). Adult Refsum's disease can be treated successfully by dietary therapy. In case of dietary treatment and BMT, assay of VLCFA and/or PHYT is important for the biochemical evaluation of these therapies.

[Zellweger syndrome, neonatal adrenoleukodystrophy or infantile Refsum's disease in a case with generalized peroxisome defect?]. / Zellweger-Syndrom, neonatale Adrenoleukodystrophie oder infantile Refsumsche Erkrankung bei einem Fall mit generalisiertem Peroxisomendefekt?

An eleven month-old boy presented clinically with craniofacial dysmorphia, severe psychomotor retardation, neurological deterioration, no response to visual and acoustic stimuli, failure to thrive, hepatomegaly and adrenal insufficiency. Specific biochemical markers for a peroxisomal deficiency disorder (Zellweger's syndrome, neonatal adrenoleukodystrophy, infantile Refsum's disease) revealed pathological results for very long chain fatty acids, phytanic acid, pristanic acid, plasmalogen biosynthesis and catalase, thus confirming the clinical diagnosis. Comparison of clinical and biochemical findings in the patient with the characteristics of the three peroxisomal deficiency disorders showed overlapping with each of these disorders, which corresponds to the current view that these three peroxisomal disorders differ only with respect to onset and severity of the clinical manifestations, but not with regard to the biochemical defects.

[Mucopolysaccharidosis V (Ullrich-Scheie syndrome) (author's transl)]. / Mukopolysaccharidose V, Morbus ullrich-Scheie.

Mucopolysaccharidosis V (Scheie's syndrome, MPS-IS) is a very rare, autosomal recessively inherited metabolic disease. The degradation of dermatan sulphate and heparan sulphate is disturbed due to alpha-L-iduronidase deficiency, leading to intracellular storage and excessive urinary secretion of these substances. The characteristic clinical features are contractures (claw-like flexion of the fingers), umbilical and inguinal herniae, corneal opacity, hepatomegaly, myocardiopathy and minor skeletal malformations. A patient with Scheie's syndrome is now reported for the first time in Austria; the results of the clinical, biochemical, chromosomal, dermatoglyphic and electron optical investigations are described and discussed.

[Adrenoleukodystrophy]. / Adrenoleukodystrophia.

7-year-old boy with adrenoleukodystrophy is presented with the typical clinical picture, biochemical findings and review of the literature. The obligate carrier status of the mother and the asymptomatic adrenoleukodystrophy of the 5-year-old brother are biochemically proved. Therapeutic regime of Lorenzo's oil has been introduced to the young brother, and the question of bone marrow transplantation is discussed.

Molecular and phenotypic characteristics of metachromatic leukodystrophy patients from Poland.

The occurrence and genotype-phenotype correlations of the eight most common mutations in the arylsulfatase A (ARSA) gene were studied in 43 unrelated Polish patients suffering from different types of metachromatic leukodystrophy (MLD). Screening for mutations p.R84Q, p.S96F, c.459+1G>A, p.I179S, p.A212V, c.1204+1G>A, p.P426L, and c.1401-1411del allowed the identification of 53.5% of the mutant alleles. In the whole investigated group of patients, mutations c.459+1G>A and p.P426L were the most frequent, 19 and 17%, respectively. The prevalence of the third most frequent mutation, i.e. p.I179S (13%), seems to be higher than that in other populations. The incidence of c.1204+1G>A was 5%, which is higher than reported earlier (2%). It seems that p.I179S and c.1204+1G>A are more prevalent in MLD patients from Poland than from other countries. In the group examined by us, mutations p.R84Q, p.S96F, p.A212V, and c.1401-1411del were not detected; thus, 46.5% of MLD alleles remained unidentified. This indicates that other, novel or already described, but rare, mutations exist in Polish population. In late infantile homozygotes for c.459+1G>A and one homozygote for c.1204+1G>A, first clinical symptom was motor deterioration. In adult homozygotes for p.P426L, the disease onset manifested as gait disturbances, followed by choreoathetotic movements, difficulties in swallowing, dysarthria, tremor, and nystagmus. In the carriers of the p.I179S mutation, the hallmark of the clinical picture was psychotic disturbances.

Diagnosis of peroxisomal disorders with neurological involvement.

Peroxisomal disorders are a group of inherited metabolic diseases caused by impairment of one or more peroxisomal functions. Ten disorders with neurological involvement have been recognized. Diagnosis and differentiation of these disorders is based on a number of important biochemical markers. For all disorders elevated values of very long chain fatty acids (VLCFA) and/or phytanic acid (PHYT) are important primary diagnostic parameters. Our results with regard to these two diagnostic markers are presented. VLCFA determined by gaschromatography in 414 samples (plasma, leukocytes or fibroblasts respectively) revealed increased values of hexacosanoic acid in 30 hemizygotes and 10 heterozygotes of adrenoleukodystrophy/adrenomyeloneuropathy and in eight infants with Zellweger disease or neonatal adrenoleukodystrophy. 15 cases with peroxisomal disorders were detected by VLCFA analysis in autopsy material. Gaschromatographic analysis of PHYT in plasma showed in some patients with Zellweger disease or neonatal adrenoleukodystrophy increase. In seven Refsum patients beside gaschromatographic demonstration of PHYT accumulation in plasma, analysis of plasma phytanyltriglycerides by thin-layer chromatography proved to be a rapid and reliable method for detection of patients and monitoring dietary treatment.

[Lipid storage diseases (lipidoses): genetic, biochemical and clinico-chemical aspects]. / Lipidspeicherkrankheiten (Lipidosen): Genetische, biochemische und klinisch-chemische Aspekte.

Lipidoses are genetic diseases due to disease-specific defects in the enzymatic catabolism of lipids, with accumulation of the respective lipid substrate in the nervous system and/or peripheral tissues. The clinical chemical diagnosis of lipidoses can be accomplished by demonstration of the enzyme defect and/or substrate accumulation in body fluids (urine, blood serum), leukocytes, cultured fibroblasts, amniotic fluid cells, or amniotic fluid, respectively. These assays are important with regard to: 1. the specific detection or exclusion of diseases, which are difficult to diagnose by their clinical presentation, 2. prenatal diagnoses, 3. detection of (clinically inconspicuous) heterozygotes (essential for individual genetic counselling), and 4. the biochemical control of dietary treatment in Refsum's disease.

Fatty acid patterns in brain, fibroblast, leukocyte and body fluid lipids in adrenoleukodystrophy.

Fatty acid patterns of certain tissue and body fluid lipids of ALD and control cases were investigated by thin layer and gas chromatography. A high percentage (55.2%) of VLFA was found in the ALD brain white matter cholesterol esters; this was mainly due to increased proportions of 26:0, 25:0, 26:1 and greater than 26 C fatty acids. The percentage of VLFA in the white matter cholesterol esters of pathological controls with myelin breakdown of different etiology was low (0.4-7.1%). In the fibroblast total lipids of an ALD patient an increase of 26:0 and 25:0 fatty acids was found. Moderately increased proportions of hexacosanoic acid were observed in the leukocyte total lipids of 2 severely affected ALD patients, but not in a slightly as well as in a possible affected case. No accumulation of VLFA was detected in cholesterol ester, triglyceride and free fatty acid fractions in the CSF of an ALD patient and in ALD serum sphingomyelin. Our results substantiate the specificity of VLFA accumulation in the white matter cholesterol esters of ALD brain and lend some further evidence to the view that a generalized impairment of VLFA metabolism seems to be present in this disease.

X-linked adrenoleukodystrophy (ALD): a novel mutation of the ALD gene in 6 members of a family presenting with 5 different phenotypes.

Fragments of the adrenoleukodystrophy (ALD) cDNA from a patient with adolescent ALD were amplified by polymerase chain reaction and subcloned. Bidirectional sequencing of the entire coding ALD gene disclosed a cytosine to guanine transversion at nucleotide 1451 in exon five, resulting in substitution of proline 484 by arginine. Five of nine siblings of the patient, comprising two cerebral ALD, one adrenomyeloneuropathy, one Addison only as well as the symptomatic mother (all accumulating very long chain fatty acids) carried this mutation, which was not found in the unaffected persons, in five unrelated ALD patients, and in twenty controls. We propose that this missense mutation generated the disease per se as well as the metabolic defect; the different phenotypes, however, must have originated by means of additional pathogenetic factors.

Simultaneous detection of the two most frequent metachromatic leukodystrophy mutations.

Metachromatic leukodystrophy (MLD) is an autosomal recessive neurometabolic disorder caused by deficiency of arylsulfatase A (ASA). To detect ASA mutations E2S609 and E8P2382, the two most frequent MLD mutations, a non-radioactive polymerase chain reaction (PCR)-based assay was developed. This assay is a multiple "mutated primer-modulated PCR restriction fragment length polymorphism". The primers related to each mutation mismatch to create an XbaI or PstI restriction site in mutation E2S609 or E8P2382, respectively. The assay was designed to give four fragments of 160, 130, 100, and 70 bp, easy to distinguish. An internal control fragment is not necessary since both primer pairs amplify different regions of the ASA gene and fragments will be obtained in all allelic possibilities. This technique produced clear-cut results when genomic DNA, isolated either from leukocytes, cultured human fibroblasts, or paraffin-embedded autopsy material, was used as template. The assay will be of help in comparative studies on the relation between MLD genotype and phenotype, a problem not yet fully understood. Since our method was shown to work also on DNA from paraffin-embedded autopsy material, genotype/phenotype studies would not be restricted to in vivo investigations but could be done also on post mortem material, thus including investigations on a large group of cases and also studies on the relation between genotype and neuropathological features.