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.     

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.     

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.     

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.     

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.     

Detection of a point mutation in sphingolipid activator protein-1 mRNA in patients with a variant form of metachromatic leukodystrophy

The lysosomal degradation of sulfatide requires the specific enzyme, arylsulfatase A, as well as a heat stable protein called sphingolipid activator protein-1 (SAP-1). While most patients with metachromatic leukodystrophy have defects in arylsulfatase A, some patients have defects in SAP-1. SAP-1 is coded for by a gene on human chromosome 10 that also codes for three other proposed SAP. Examination of the cDNA from two siblings with SAP-1 deficiency revealed a point mutation of nucleotide #650 (counting from the initiation ATG) which is in the SAP-1 coding domain. This C to T transition changed the codon from threonine (ACC) to one coding for isoleucine (ATC). This eliminated the only glycosylation site in mature SAP-1 and could explain the findings made at the protein level.     

Identification of a splice-site mutation in the aldolase B gene from an individual with hereditary fructose intolerance.

Hereditary fructose intolerance (HFI) is a potentially fatal autosomal recessive disease of carbohydrate metabolism. HFI patients exhibit a deficiency of fructose 1-phosphate aldolase (aldolase B), the isozyme expressed in tissues that metabolize fructose. The eight protein-coding exons, including splicing signals, of the aldolase B gene from one HFI patient were amplified by PCR. Dot-blot hybridization of the amplified DNA with allele-specific oligonucleotide (ASO) probes revealed a previously described A149P mutation in one allele from the proband. The mutation in the other allele was identified by direct sequencing of the double-stranded PCR-amplified material from the proband. The nucleotide sequence of exon 9 revealed a 7-base deletion/1-base insertion (delta 7 + 1) at the 3' splice site of intron 8 in one allele. This mutation was confirmed by cloning PCR-amplified exon 9 of the proband and determining the sequence of each allele separately. ASO analysis of 18 family members confirmed the Mendelian inheritance of both mutant alleles. The implications of this unique splice-site mutation in HFI are discussed.     

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.     

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.     

Somatic mosaicism for a newly identified splice-site mutation in a patient with adenosine deaminase-deficient immunodeficiency and spontaneous clinical recovery.

Absent or severely reduced adenosine deaminase (ADA) activity produces inherited immunodeficiency of varying severity, with defects of both cellular and humoral immunity. We report somatic mosaicism as the basis for a delayed presentation and unusual course of a currently healthy young adult receiving no therapy. He was diagnosed at age 2 1/2 years because of life-threatening pneumonia, recurrent infections, failure of normal growth, and lymphopenia, but he retained significant cellular immune function. A fibroblast cell line and a B cell line, established at diagnosis, lacked ADA activity and were heteroallelic for splice-donor-site mutation in IVS 1 (+1GT-->CT) and a missense mutation (Arg101Gln). All clones (17/17) isolated from the B cell mRNA carried the missense mutation, indicating that the allele with the splice-site mutation produced unstable mRNA. In striking contrast, a B cell line established at age 16 years expressed 50% of normal ADA; 50% of ADA mRNA had normal sequence, and 50% had the missense mutation. Genomic DNA contained the missense mutation but not the splice-site mutation. All three cell lines were identical for multiple polymorphic markers and the presence of a Y chromosome. In vivo somatic mosaicism was demonstrated in genomic DNA from peripheral blood cells obtained at 16 years of age, in that less than half the DNA carried the splice-site mutation (P < .002, vs. original B cell line). Consistent with mosaicism, erythrocyte content of the toxic metabolite deoxyATP was only minimally elevated. Somatic mosaicism could have arisen either by somatic mutation or by reversion at the site of mutation. Selection in vivo for ADA normal hematopoietic cells may have played a role in the return to normal health, in the absence of therapy.     

Accumulation of lysosulfatide (sulfogalactosylsphingosine) in tissues of a boy with metachromatic leukodystrophy

Abnormal accumulation of lysosulfatide (sulfogalactosylsphingosine) was evident in autopsied tissues from a boy with late-infantile metachromatic leukodystrophy. The concentration was high in the cerebral white matter, spinal cord and sciatic nerve (116-787 pmol/mg protein) and low in the cerebral gray matter, kidney and liver (4-40 pmol/mg protein). As is the case with galactosylsphingosine, lysosulfatide inhibited cytochrome c oxidase activity, in a dose-dependent manner. Judging from the tissue distribution of the accumulated lysosulfatide and because of the cytotoxicity, the lysosulfatide presumably explains the demyelination seen in the nervous tissues of patients with metachromatic leukodystrophy.