John S.Harvey, William F.Carey, Paul V.Nelson and C.Phillip Morris*. Department of Chemical Pathology, Women's and Children's Hospital, 72 King William ...
© 1994 Oxford University Press
Human Molecular Genetics, 1994, Vol. 3, No. 1 207
Metachromatic leukodystrophy: a nonsense mutation (Q486X) in the arylsulphatase A (ARSA) gene John S.Harvey, William F.Carey, Paul V.Nelson and C.Phillip Morris* Department of Chemical Pathology, Women's and Children's Hospital, 72 King William Road, North Adelaide, SA 5006, Australia
Received September 28, 1993; Revised and Accepted November 19, 1993
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. The patient in this study presented with late-infantile MLD suggesting that the Q4g6X mutation and an as yet unidentified mutant allele are associated with the development of the most severe MLD clinical phenotype. The Q4g6X mutation is expected to produce a truncated protein missing 22 of the 507 amino acids that make up the ARSA precursor, it is most likely that such a mutant protein would be either unstable or lacking in enzyme activity. This is consistent with the virtual absence
* To whom correspondence should be addressed
of ARSA activity in this patient's leukocytes or skin fibroblasts and the clinical presentation of the patient. ACKNOWLEDGMENTS We wish to thank G.J.HewittfromWagga Base Hospital forreferralof the patient. This work was supported by the Women's and Children's Hospital Research Foundation.
REFERENCES 1. Kolodny,E.H. (1989) In Scriver.C.R., Beaudet.A.L., Sly.W.S. and Valle,D. (eds), The Metabolic Basis ofInherited Disease. 6th Ed. McGraw-Hill, New York. pp. 1721-1750. 2. Nelson,P.V., Carey.W.F. and Morris.C.P. (1991) Hum. Genet. 87, 87-88. 3. KreysingJ., von Figura.K. and Gieselmann.V. (1990) Eur. J. Biochem. 191, 627-631. 4. Harvey,J.S., Nelson.P.V., Robertson.E.F., Carey.W.F. and Morris.C.P. (1993) Hum. Mut. 2, 261-267. 5. Murray,V. (1989) Nucleic Adds Res. 17, 8889.
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4
Figure 1. SSCP analysis of Exon 8. Lane 1: MLD patent. Lanes 2 - 4 : Controls. A band in lane 1 with altered mobility is arrowed.
1 2
Filter A:
Filter B:
3
f 7 V
Normal Q486
Mutant X486
Figure 2. Detection of the (J^^X mutation using normal (Qwtf, filter A) and mutant (X486, filter B) allele specific oligonucleotides. Lanes 1-2: Controls, Lane 3: MLD patient who is heterozygous
