Determination of the molecular defect of caprine N-acetylglucosamine 6-sulfatase deficiency

Caprine N-acetylglucosamine 6-sulfatase (G6S) deficiency is the only animal analog of Sanfilippo syndrome (type D). The goat with this mucopolysaccharidousis disorder (MPS III D) demonstrated delayed motor development and growth retardation but reached sexual maturity before dying suddenly at 19 mo. Histochemical and biochemical analysis of the liver showed glycosaminoglycan storage and there was GM{sub 3} ganglioside accumulation in the brain. Towards further development of this animal model for treatment strategies, we have cloned the caprine G6S gene, determined the nature of the gene defect in caprine MPS III D and compared the goat sequence to the human sequence. The human and caprine sequences show an overall sequence similarity of about 90% in the coding region. The 5{prime}-coding region is very GC-rich in both the human and caprine G6S. One striking difference between the human and caprine genes is the presence of a GCC repeat in the goat resulting in insertion of 6 prolines and a leucine in the signal peptide. This proline-rich stretch was confirmed by amplifying and sequencing the same cDNA segment from other goats. Additionally, this region was examined in bovine cDNA and found to contain 4 prolines and 2 leucines. The mRNA for G6S consists ofmore » two species of approximately 4.0 and 4.2 kb with a coding region of 1.6 kb. For mutation analysis a series of primers was designed to cover the entire G6S coding region. Amplicons from RT-PCR on normal and affected goat total RNA were produced and sequenced. A single base substitution, T for C, was found in the 5{prime} region of the coding sequence of the affected animals that creates a stop codon. This mutation introduces an Alu I restriction site. PCR primers designed to amplify a short segment of genomic DNA encompassing the mutation have been used to identify putative carriers and develop a caprine Sanfilippo III D carrier colony.« less