SIMULTANEOUS DETECTION OF THE 2 MOST FREQUENT METACHROMATIC LEUKODYSTROPHY MUTATIONS

Metachromatic leukodystrophy (MLD) is an autosomal recessive neurometa bolic disorder caused by deficiency of arylsulfatase A (ASA). To detec t ASA mutations E2S609 and E8P2382, the two most frequent MLD mutation s, a non-radioactive polymerase chain reaction (PCR)-based assay was d eveloped. This assay is a multiple ''mutated primer-modulated PCR rest riction fragment length polymorphism''. The primers related to each mu tation mismatch to create an XbaI or PstI 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 interna l control fragment is not necessary since both primer pairs amplify di fferent 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 fibroblas ts, or paraffin-embedded autopsy material, was used as template. The a ssay will be of help in comparative studies on the relation between ML D genotype and phenotype, a problem not yet fully understood. Since ou r method was shown to work also on DNA from paraffin-embedded autopsy material, genotype/phenotype studies would not be restricted to in viv o 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.