The mechanism for a 33-nucleotide insertion in mRNA causing sphingolipid activator protein (SAP-1)-deficient metachromatic leukodystrophy.

Metachromatic leukodystrophy is a severe autosomal recessive disorder caused by accumulation of sulfatide resulting from deficient lysosomal degradation. While most patients have mutations in the lysosomal enzyme arylsulfatase A, some patients have mutations in a required heat stable sphingolipid activator protein, we call SAP-1. One patient with SAP-1 deficiency was previously demonstrated to have a 33-nucleotide insertion in her mRNA. This resulted in the production of mature SAP-1 with 11 extra amino acids, which was unstable during intracellular processing. In this manuscript we demonstrate that the 33 nucleotides are present near the middle of a 4-kb intron, and that a single base change, c to a, in the second position preceding the 33-nucleotide insertion, coupled with the presence of a string of pyrimidines immediately upstream from this change, creates a new 3' splice junction. The presence of a string of pyrimidines within the 33-nucleotide insertion, which has three cag trinucleotides near the 3' end, leads to alternative splicing in normal people as found in this laboratory and by others. The insertion region is followed by a gt dinucleotide that is spliced to a typical 3' consensus sequence. The single nucleotide change, c to a, was confirmed by identifying normal and mutant sequence in the consanguineous parents and a sister, previously identified as a carrier of this disorder.