A SINGLE-BASE-PAIR DELETION IN THE BETA-GLUCURONIDASE GENE ACCOUNTS FOR THE PHENOTYPE OF MURINE MUCOPOLYSACCHARIDOSIS TYPE-VII

Murine mucopolysaccharidosis type VII is a heritable disease caused by a spontaneous mutation, gus(mps), closely linked to the beta-glucuron idase structural gene on chromosome 5. Mice homozygous for the mutatio n have a >200-fold decrease in beta-glucuronidase mRNA levels and virt ually no enzyme activity detectable by a sensitive fluorometric assay. Approximately 20 kb of genomic DNA containing the beta-glucuronidase gene Gus and >2 kb of 5' and 3' flanking sequences were cloned from bo th a gus(mps)/gus(mps) mouse and a +/+ mouse of the progenitor strain. Restriction enzyme digests containing DNA fragments 20-400 bp in leng th were generated from each of the two Gus alleles and then compared b y using nondenaturing polyacrylamide DNA-sequencing gels. This method rapidly identified a large number of restriction sites and was sensiti ve enough to detect a restriction fragment length variation resulting from a 1-bp deletion in the gus(mps) allele. DNA-sequence analysis of the mutant genomic fragment showed that the 1-bp deletion created a fr ameshift mutation within exon 10. Insertion of the deleted nucleotide by oligonucleotide site-directed mutagenesis restored function to the corrected mutant gene when transfected into gus(mps)/gus(mps) fibrobla sts. We concluded that the frameshift mutation, which introduces a pre mature stop codon at codon 497 in exon 10, accounts for the molecular, biochemical, and pathological abnormalities associated with the gus(m ps) phenotype.