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.     

Induction of tolerance to human arylsulfatase A in a mouse model of metachromatic leukodystrophy

A deficiency of arylsulfatase A (ASA) causes metachromatic leukodystrophy (MLD), a lysosomal storage disorder characterized by accumulation of sulfatide, a severe neurological phenotype and early death. The efficacy of enzyme replacement therapy (ERT) has previously been determined in ASA knockout (ASA-/-) mice representing the only available animal model for MLD. Repeated intravenous injection of human ASA (hASA) improved the nervous system pathology and function, but also elicited a progressive humoral immune response leading to treatment resistance, anaphylactic reactions, and high mortality. In contrast to ASA-/- mice, most MLD patients express mutant hASA which may entail immunological tolerance to substituted wildtype hASA and thus protect from immunological complications. To test this notion, a cysteine-to-serine substitution was introduced into the active site of the hASA and the resulting inactive hASA-C69S variant was constitutively expressed in ASA-/- mice. Mice with sub-to supranormal levels of mutant hASA expression were analyzed. All mice, including those showing transgene expression below the limit of detection, were immunologically unresponsive to injected hASA. More than 100-fold overexpression did not induce an overt new phenotype except occasional intralysosomal deposition of minor amounts of glycogen in hepatocytes. Furthermore, long-term, low-dose ERT reduced sulfatide storage in peripheral tissues and the central nervous system indicating that high levels of extracellular mutant hASA do not prevent cellular uptake and lysosomal targeting of substituted wildtype hASA. Due to the tolerance to hASA and maintenance of the MLD-like phenotype, the novel transgenic strain may be particularly advantageous to assess the benefit and risk of long-term ERT.     

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.     

Two new arylsulfatase A (ARSA) mutations in a juvenile metachromatic leukodystrophy (MLD) patient.

Fragments of the arylsulfatase A (ARSA) gene from a patient with juvenile-onset metachromatic leukodystrophy (MLD) were amplified by PCR and ligated into MP13 cloning vectors. Clones hybridizing with cDNA for human ARSA were selected, examined for appropriate size inserts, and used to prepare single-stranded phage DNA. Examination of the entire coding and most of the intronic sequence revealed two putative disease-related mutations. One, a point mutation in exon 3, resulted in the substitution of isoleucine by serine. Introduction of this alteration into the normal ARSA cDNA sequence resulted in a substantial decrease in ARSA activity on transient expression in cultured baby hamster kidney cells. About 5% of the control expression was observed, suggesting a small residual activity in the mutated ARSA. The second mutation, a G-to-A transition, occurred in the other allele and resulted in an altered splice-recognition sequence between exon 7 and the following intron. The mutation also resulted in the loss of a restriction site. Apparently normal levels of mRNA were generated from this allele, but no ARSA activity or immuno-cross-reactive material could be detected. A collection of DNA samples from known or suspected MLD patients, members of their families, and normal controls was screened for these mutations. Four additional individuals carrying each of the mutations were found among the nearly 100 MLD patients in the sample. Gene segregation in the original patient's family was consistent with available clinical and biochemical data. No individuals homozygous for either of these two mutations were identified. However, combinations with other MLD mutations suggest that the point mutation in exon 3 does result in some residual enzyme activity and is associated with late-onset forms of the disease. The splice-site mutation following exon 7 produces late-infantile MLD when combined with other enzyme-null mutations, implying that it is completely silent enzymatically.     

An adult-type metachromatic leukodystrophy caused by substitution of serine for glycine-122 in arylsulfatase A

Metachromatic leukodystrophy (MLD) is a lysosomal storage disease with autosomal recessive inheritance caused by a deficiency of the enzyme arylsulfatase A (ASA). We have identified a new mutation in the ASA gene of a patient with adult-type MLD. In this mutation, the glycine at position 122, a highly conserved residue in the AS gene family, was replaced by serine. In a transient expression study, COS cells transfected with the mutant cDNA carrying ¹²²GlySer did not show an increase of ASA activity and produced little material immunoreactive to an anti-ASA antibody, despite normal mRNA levels.     

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.     

An 11-bp deletion in the arylsulfatase A gene of a patient with late infantile metachromatic leukodystrophy

Summary Metachromatic leukodystrophy is a lysosomal storage disorder caused by the deficiency of arylsulfatase A. Examination of the arylsulfatase A gene in a patient suffering from late infantile metachromatic leukodystrophy revealed an 11-bp deletion in exon 8. Although this allele produces normal amounts of ASA mRNA, no arylsulfatase A cross-reacting material could be detected in cultured fibroblasts from the patient. The patient was found to be a compound heterozygote, the other allele is also known to generate no ASA polypeptides. This patient is another example where absence of ASA polypeptides correlates with the severe late infantile form of metachromatic leukodystrophy.     

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.     

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.     

Compound heterozygosity for metachromatic leukodystrophy and arylsulfatase A pseudodeficiency alleles is not associated with progressive neurological disease

Several allelic mutations at the arylsulfatase A (ASA) locus cause substantial deficiencies of this lysosomal enzyme. Depending on the genetically determined degree of the deficiency, the clinical outcome may be very different—either metachromatic leukodystrophy (MLD), a lethal lysosomal storage disorder affecting the nervous system, or, more frequently, the so-called pseudodeficiency (PD), which has no apparent clinical consequence. Because of compound heterozygosity for MLD and PD, 1/1,000 individuals in the population have low residual enzyme activities, which are intermediate between those of MLD patients and those of PD homozygous normal individuals. In order to assess whether PD/MLD compound heterozygotes bear a health risk, we examined clinically and biochemically 16 individuals with this genotype. Of these subjects, two had neurological symptoms and two showed lesions, without clinical symptoms, in magnetic resonance imaging of the brain. None of these symptoms was progressive, nor did they resemble those of MLD. Nerve conduction velocities were normal in these probands, and they secreted only low amounts of sulfatide in the urine. We conclude that the observed neurological symptoms are unrelated to the ASA genotype and that PD/MLD compound heterozygotes are not at an increased risk for developing progressive nervous system diseases.     

A 9-bp deletion (2320del9) on the background of the arylsulfatase A pseudodeficiency allele in a metachromatic leukodystrophy patient and in a patient with nonprogressive neurological symptoms

A 9-bp deletion (2320del9) was detected in the arylsulfatase A genes of a patient with late infantile metachromatic leukodystrophy and of a patient with nonprogressive neurological symptoms and very low arylsulfatase A activity. Both patients are heterozygous for the deletion, which involves codons 406–408 and causes loss of a Ser-Asp-Thr tract in the predicted protein. In both patients the 9-bp deletion lies in a pseudodeficiency allele. The patient with metachromatic leukodystrophy carries the common 459 + 1G > A mutation in the other allele. The other patient is homozygous for the pseudodeficiency allele, and consequently is a compound heterozygote for a metachromatic leukodystrophy allele and a pseudodeficiency allele. We hypothesize that the compound heterozygosity predisposes to the development of nonprogressive neurological symptoms in the presence of additional, still unknown, genetic or nongenetic factors. Received: 18 April 1997 / Accepted: 16 August 1997     

Very low arylsulfatase A and cerebroside sulfatase activities in leukocytes of healthy members of metachromatic leukodystrophy family.

Very low levels of arylsulfatase A (ASA) have been found in the leukocytes of healthy members of a metachromatic leukodystrophy (MLD) family. The cerebroside sulfate sulfatase (CSS) activities in the same individuals are about 10% of the control level. Arguments favoring a dominant mutation different from that of classical MLD are presented. This report reinforces the relationship between the two enzymatic activities.     

Somatic cell hybridization studies on the genetic regulation and allelic mutations in metachromatic leukodystrophy

Summary Metachromatic leukodystrophy is a hereditary neurodegenerative disease associated with deficient arylsulfatase A activity. Clinical variants differ in onset times and severity of the disease but each breeds true within families. Somatic cell hybridization techniques were used to clarify the genetic relationship among these mutants. Hybrid clones isolated with a nonselective method from fusing fibroblasts of an infantile and a juvenile variant did not show complementation of arylsulfatase A activity. Hence, these clinical variants are allelic mutants.Previous somatic cell hybridization studies suggested that arylsulfatase A-deficiency is a dominant phenotype, in contrast to its apparent recessive mode of inheritance. To resolve this discrepancy, hybrid clones from fusing normal and arylsulfatase A-deficient fibroblasts were isolated nonselectively. They continued to express arylsulfatase A activity. Hence, even in vitro, arylsulfatase A-deficiency remains as a recessive phenotype.     

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.     

Prevalence of common mutations in the arylsulphatase A gene in metachromatic leukodystrophy patients diagnosed in Britain

The frequency of two common disease-associated mutations in the arylsulphatase A (ASA) gene, and of a mutation causing ASA pseudodeficiency, have been established in metachromatic leukodystrophy patients diagnosed in our laboratory. A total of 37 mutant genes have been analysed. The GA change destroying the splice donor site of exon 2 is generally associated with more severe disease and was found in 43.2% of mutant ASA genes. The CT transition causing a proline to leucine substitution at position 426 in exon 8 (P426L) is associated with later onset disease, and was found in 16.2% of mutant genes. The AG transition leading to loss of a polyadenylation signal associated with ASA pseudodeficiency was present at a frequency of 7.5% in the patients and heterozygotes studied.     

Molecular screening of the major mutations in the ARSA gene in patients with metachromatic leukodystrophy

Metachromatic leukodystrophy (MLD) is an inherited storage disease caused by deficiency of arylsulfatase A (ARSA). Molecular analysis of the major mutations in the ARSA gene was performed in 10 Ukrainian patients (from 9 families) with MLD. According to the age of onset, late infantile MLD was identified in 3 patients, juvenile MLD in 5 patients, and adult MLD in 2 patients (sibs), respectively. The ARSA activity in the patients was 2-26 nmol/h/mg protein (the normal activity has been established in our laboratory as 111.9 +/- 7.1 nmol/h/mg protein). No correlation between enzyme activity and a clinical course of disease was revealed. The IVS2 + 1 mutation was found at 2 of 20 alleles (in a patient with late infantile form) and the P426L mutation was found at 2 of 20 alleles (in two patients with juvenile form). Thus, the total frequency of these two major mutations in the ARSA gene is 20% in Ukrainian MLD patients.     

Multiple mutations are responsible for the high frequency of metachromatic leukodystrophy in a small geographic area.

Metachromatic leukodystrophy is a lysosomal storage disorder caused by the deficiency of arylsulfatase A. The disease occurs panethnically, with an estimated frequency of 1/40,000. Metachromatic leukodystrophy was found to be more frequent among Arabs living in two restricted areas in Israel. Ten families with affected children have been found, three in the Jerusalem region and seven in a small area in lower Galilee. Whereas all patients from the Jerusalem region are homozygous for a frequent mutant arylsulfatase A allele, five different mutations were found in the families from lower Galilee. In patients of Muslim Arab origin, we have found a G86-->D, a S96-->L, and a Q190-->H substitution. Two different defective arylsulfatase A alleles, characterized by a T274-->M and a R370-->W substitution, respectively, have been found among the Christian Arab patients. All mutations were introduced into the wild-type arylsulfatase A cDNA. No enzyme activity could be expressed from the mutagenized cDNAs after transfection into heterologous cells. In all instances, the patients were found to be homozygous for the mutations, and four of the five mutations occurred on different haplotypes. The clustering of this rare lysosomal storage disease in a small geographic area usually suggests a founder effect, so the finding of five different mutations is surprising.