Metachromatic leukodystrophy: a nonsense mutation (Q486X) in the arylsulphatase A (ARSA) gene

Abstract

Metachromatic Leukodystrophy (MLD) is an autosomal recessive lysosomal storage disorder characterised by defective catabolism of myelin and the accumulation of cerebroside sulphate in neural tissue. The disorder is caused by a deficiency of the lysosomal hydrolase, arylsulphatase A (ARSA). MLD patients present with a broad spectrum of clinical phenotypes, the disorder has therefore been divided into three broad subtypes based on the age of onset of clinical symptoms. Late-infantile MLD is typically diagnosed before the age of 2 years, juvenile between ages 3 and 16 and adult MLD which is diagnosed after puberty (1). Biochemical diagnosis of MLD is complicated by the existence of an ARSA pseudodeficiency allele, which causes the loss of the major mRNA species (2.1 kb) of ARSA and a concomitant 90% reduction in the level of ARSA activity. Approximately 1% of the normal population is homozygous for this allele with no apparent clinical sequelae (2). The 507 amino acid ARSA polypeptide is encoded by a compact 3.2 kb gene containing 8 exons (3). Each of the ARSA exons and intron boundaries were PCR amplified and subjected to single-stranded conformation polymorphism analysis (SSCP) to detect sequence alterations (4). Exons exhibiting alterations in electrophoretic mobility on SSCP gels were sequenced using a modified linear based PCR sequencing protocol (5). SSCP analysis of the ARSA gene from a late-infantile MLD patient revealed a shift in the mobility of a band derived from exon v m (Figure 1). Sequence analysis of exon 8 revealed a heterozygous C to T transition at nucleotide number 1458 of the ARSA cDNA resulting in the alteration of a glutamine codon (CAG) at position 486 to a stop codon (TAG), (Q486X). No other sequence changes have yet been observed in this patient. Allele specific oligonucleotides were designed to detect the normal (5'-CGCCCTGCAGATCTGCTG-3') and mutant (5'-CGCCCTGTAGATCTGCTG-3') sequences Of Q486X (Figure 2) on Southern blotted filters containing exon 8 PCR products. After hybridisation filters were washed twice in 4xSSC for 10 minutes and once at 60°C in 2 xSSC, 0.1 % SDS (4). The patient's mother was found to be heterozygous for Q ggX (her father was unavailable for testing), however the mutation was not found in 27 other MLD patients, 21 ARSA pseudodeficient or 42 normal individuals.