Sulfatide excreting heterozygous carrier of juvenile metachromatic leukodystrophy or asymptomatic patient of adult metachromatic leukodystrophy.

In a family with juvenile metachromatic leukodystrophy (sulfatide lipidosis) 2 patients showed residual arysulfatase A activities of 5--6%. The patients' healthy father was characterized biochemically by a 39% normal activity of leukocyte plus plasma arylsulfatase A. The father was further characterized by a high sulfatide excretion (0.2--0.5 mg/I urine) and, paradoxically, by a normal sulfatide degrading enzyme activity in vitro. This special carrier is suspected to be heterozygous for a) arylsulfatase A deficiency and b) arylsulfatase A (sulfatidase) lability. This presumed additional genetic defect could be the cause of the sulfatide excretion which, in turn, would be a sign of the preclinical stage of an exceptional form of adult metachromatic leukodystrophy. The normal sulfatidase activity seems to be due to an in vitro effect.     

Cerebroside sulfatase activator deficiency induced metachromatic leukodystrophy

Two siblings of consanguineous parents had presented with a variety of findings indicative of juvenile metachromatic leukodystrophy (MLD). However, instead of the expected profound deficiency of arylsulfatase A (ARS A), their enzyme levels were about half-normal, and enzyme from fibroblasts had properties identical with the properties of enzyme from normal fibroblasts. Nevertheless, the hydrolysis of cerebroside sulfate by growing fibroblasts was markedly attenuated. Supplementation of the fibroblasts with cerebroside sulfatase activator normalized the response in the loading test. These results imply that the fibroblasts, and by extension the patients, are deficient in activator. Although the defective catabolism of cerebroside sulfate and the clinical manifestations in these patients mimic MLD, the molecular basis is distinct from the classical forms of the disorder.     

Mutations in the arylsulfatase A pseudodeficiency allele causing metachromatic leukodystrophy.

We identified a patient suffering from late infantile metachromatic leukodystrophy who genetically seemed to be homozygous for the mutations signifying the arylsulfatase A pseudodeficiency allele. Homozygosity for the pseudodeficiency allele is associated with low arylsulfatase A activity but does not cause a disease. Analysis of the arylsulfatase A gene in this patient revealed a C----T transition in exon 2, causing a Ser 96----Phe substitution in addition to the sequence alterations causing arylsulfatase A pseudodeficiency. Although this mutation was found only in 1 of 78 metachromatic leukodystrophy patients tested, five more patients were identified who seemed hetero- or homozygous for the pseudodeficiency allele. The existence of nonfunctional arylsulfatase A alleles derived from the pseudodeficiency allele calls for caution when the diagnosis of arylsulfatase A pseudodeficiency is based solely on the identification of the mutations characterizing the pseudodeficiency allele.     

Heterogeneity in late-onset metachromatic leukodystrophy. Effect of inhibitors of cysteine proteinases.

The synthesis of arylsulfatase A polypeptides was followed in fibroblasts from 11 patients with late-onset forms of metachromatic leukodystrophy. In 10 cell lines, the apparent rate of synthesis was 20%-70% as measured by the amount of [35S]arylsulfatase A secreted in the presence of 10 mM NH4Cl. The specific activity of the secreted arylsulfatase A was normal. The residual activity of arylsulfatase A was below 10% except for one cell line in which it was 20%. The activity of arylsulfatase A and the degradation of sulfatides was partially restored in these fibroblast lines by treatment with irreversible (peptidyl diazomethyl ketones) or competitive (leupeptin) inhibitors of cysteine proteinases. Thus, the mutation(s) in these cell lines led to the synthesis of arylsulfatase. A polypeptides with increased susceptibility to cysteine proteinases. Multiple allelic mutations within this group of late-onset metachromatic leukodystrophy were suggested by the clinical heterogeneity, the variability of the residual activity, and in the response to inhibitors of cysteine proteinases. In fibroblasts from one patient, the apparent rate of synthesis of arylsulfatase A was less than 5%. Furthermore, inhibitors of cysteine proteinases were without effect, suggesting that the mutation in this patient is different from the others.     

Neuropsychological deficits in obligatory heterozygotes for metachromatic leukodystrophy

Summary Two groups of heterozygotes, one for metachromatic leukodystrophy (MLD) and the other for Tay-Sachs disease, were given a battery of neuropsychological tests, a standard neurological examination, and an EEG. Neurological and EEG findings were unremarkable for both groups. The MLD heterozygotes showed deficits in the neuropsychological tests involving spatial or constructional components, but not in tests involving language skills. The Tay-Sachs heterozygotes showed no consistent deficit on any component of the neuropsychological tests.     

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 anXbaI orPstI 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.     

Staining of sulphatides in metachromatic leukodystrophy with Alcian blue at high salt concentrations.

Alcian blue combines with purified sulphatide in 1.OM magnesium chloride. In tissue sections from patients with metachromatic leukodystrophy, sulphatide is stained by Alcian blue in 0.8 M magnesium chloride, and the staining can be abolished by prior treatment with chloroform and methanol. The simplicity of the technique, its specificity and ease of interpretation recommend Alcian blue staining at high salt concentrations as a routine method in the diagnosis of metachromatic leukodystrophy.     

Human urinary sulfatides in patients with sulfatidosis (metachromatic leukodystrophy)

The excretion of sulfatides in human urine was studied. 24-hr urine collections were filtered. Urinary glycolipids were extracted from the filter paper and fractionated on diethylaminoethyl cellulose and silicic acid columns, and by thin-layer chromatography. Fatty acids and long-chain bases were analyzed by gas-liquid chromatography of the corresponding esters and aldehydes. Glycosyl ceramide concentration was determined by gas-liquid chromatography of the trimethylsilyl ethers of the methyl glycosides. Normal females were found to excrete larger amounts of dihexosyl ceramides than males. Sulfatides were detected in all urine specimens. In sulfatidosis, a hereditary sulfatide storage disorder known as metachromatic leukcdystrophy, a large increase in sulfatide was readily apparent on a thin-layer chromatogram of the crude lipid extract. On comparing samples from normal individuals and patients with sulfatidosis, urinary sulfatide composition was remarkably similar to that previously reported in the kidney, including differences in fatty acid pattern. The determination of urinary sulfatides was a valuable confirmation of the deficiency in arylsulfatase A activity characteristic of sulfatidosis.     

High residual arylsulfatase A (ARSA) activity in a patient with late-infantile metachromatic leukodystrophy.

We identified a patient suffering from late-infantile metachromatic leukodystrophy (MLD) who has a residual arylsulfatase A (ARSA) activity of about 10%. Fibroblasts of the patient show significant sulfatide degradation activity exceeding that of adult MLD patients. Analysis of the ARSA gene in this patient revealed heterozygosity for two new mutant alleles: in one allele, deletion of C 447 in exon 2 leads to a frameshift and to a premature stop codon at amino acid position 105; in the second allele, a G-->A transition in exon 5 causes a Gly309-->Ser substitution. Transient expression of the mutant Ser309-ARSA resulted in only 13% enzyme activity of that observed in cells expressing normal ARSA. The mutant ARSA is correctly targeted to the lysosomes but is unstable. These findings are in contrast to previous results showing that the late-infantile type of MLD is always associated with the complete absence of ARSA activity. The expression of the mutant ARSA protein may be influenced by particular features of oligodendrocytes, such that the level of mutant enzyme is lower in these cells than in others.     

Identification of a mutation in the arylsulfatase A gene of a patient with adult-type metachromatic leukodystrophy.

To analyze the genetic abnormality in a Japanese patient with adult-type metachromatic leukodystrophy (MLD), we first elucidated the genomic organization of the human arylsulfatase A (ASA) gene and then compared the nucleotide sequences of exons and splice junctions of the mutant ASA gene to those of a normal control. We have identified a new mutation, a G-to-A transition in exon 2, which results in amino acid substitution of Asp for 99Gly. In a transient expression study, COS cells transfected with the mutant cDNA carrying 99Gly----Asp did not show an increase of ASA activity, which confirms that the mutation is a cause of adult-type MLD.     

Defective oligomerization of arylsulfatase a as a cause of its instability in lysosomes and metachromatic leukodystrophy.

In one of the most common mutations causing metachromatic leukodystrophy, the P426L-allele of arylsulfatase A (ASA), the deficiency of ASA results from its instability in lysosomes. Inhibition of lysosomal cysteine proteinases protects the P426L-ASA and restores the sulfatide catabolism in fibroblasts of the patients. P426L-ASA, but not wild type ASA, was cleaved by purified cathepsin L at threonine 421 yielding 54- and 9-kDa fragments. X-ray crystallography at 2.5-A resolution showed that cleavage is not due to a difference in the protein fold that would expose the peptide bond following threonine 421 to proteases. Octamerization, which depends on protonation of Glu-424, was impaired for P426L-ASA. The mutation lowers the pH for the octamer/dimer equilibrium by 0.6 pH units from pH 5.8 to 5.2. A second oligomerization mutant (ASA-A464R) was generated that failed to octamerize even at pH 4.8. A464R-ASA was degraded in lysosomes to catalytically active 54-kDa intermediate. In cathepsin L-deficient fibroblasts, degradation of P426L-ASA and A464R-ASA to the 54-kDa fragment was reduced, while further degradation was blocked. This indicates that defective oligomerization of ASA allows degradation of ASA to a catalytically active 54-kDa intermediate by lysosomal cysteine proteinases, including cathepsin L. Further degradation of the 54-kDa intermediate critically depends on cathepsin L and is modified by the structure of the 9-kDa cleavage product.     

Studies in metachromatic leukodystrophy: XV. Purification of normal and mutant arylsulfatase A from human liver

In this report we describe a method to purify both normal and abnormal (inactive) arylsulfatase A. The abnormal enzyme protein was isolated both from cases of late infantile and early juvenile forms of metachromatic leukodystrophy. Conventional protein isolation methods reported earlier were followed by size exclusion high-performance liquid chromatography in the final purification stages. Both the mutant enzyme and the normal enzyme had the same HPLC elution behavior. They thus appeared to self-associate in a similar pH-dependent fashion. Both could be followed by their reaction to a rabbit antibody to normal human arylsulfatase A. The amount of homogenous protein obtained from about 500 grams of liver was 300-400 micrograms.