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 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 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.