A Single-Base-Pair Deletion in the β-Glucuronidase Gene Accounts for the Phenotype of Murine Mucopolysaccharidosis Type VII

Murine mucopolysaccharidosis type VII is a heritable disease caused by a spontaneous mutation, gusmps, closely linked to the β-glucuronidase structural gene on chromosome 5. Mice homozygous for the mutation have a >200-fold decrease in β-glucuronidase mRNA levels and virtually no enzyme activity detectable by a sensitive fluorometric assay. Approximately 20 kb of genomic DNA containing the β-glucuronidase gene Gus and >2 kb of 5' and 3' flanking sequences were cloned from both a gusmps/gusmpsmouse and a +/+ mouse of the progenitor strain. Restriction enzyme digests containing DNA fragments 20-400 bp in length were generated from each of the two Gus alleles and then compared by using nondenaturing polyacrylamide DNA-sequencing gels. This method rapidly identified a large number of restriction sites and was sensitive enough to detect a restriction fragment length variation resulting from a 1-bp deletion in the gusmpsallele. DNA-sequence analysis of the mutant genomic fragment showed that the 1-bp deletion created a frameshift mutation within exon 10. Insertion of the deleted nucleotide by oligonucleotide site-directed mutagenesis restored function to the corrected mutant gene when transfected into gusmps/gusmpsfibroblasts. We concluded that the frameshift mutation, which introduces a premature stop codon at codon 497 in exon 10, accounts for the molecular, biochemical, and pathological abnormalities associated with the gusmpsphenotype.