Molecular diagnosis of mucopolysaccharidosis type II (Hunter syndrome) by automated sequencing and computer-assisted interpretation: Toward mutation mapping of the iduronate-2-sulfatase gene

Virtually all mutations causing Hunter syndrome (mucopolysaccharidosis type II) are expected to be new mutations. Therefore, as a means of molecular diagnosis, we developed a rapid method to sequence the entire iduronate-2- sulfatase (IDS) coding region. PCR amplicons representing the IDS cDNA were sequenced with an automatic instrument, and output was analyzed by computer- assisted interpretation of tracings, using Staden programs on a Sun computer. Mutations were found in 10 of 11 patients studied. Unique missense mutations were identified in five patients: H229Y (685C→T, severe phenotype); P358R (1073C→G, severe); R468W (1402C→T, mild); P469H (1406C→A, mild); and Y523C (1568A→G, mild). Nonsense mutations were identified in two patients: R172X (514C→T, severe) and Q389X (1165C→T, severe). Two other patients with severe disease had insertions of 1 and 14 bp, in exons 3 and 6, respectively. In another patient with severe disease, the predominant (>95%) IDS message resulted from aberrant splicing, which skipped exon 3. In this last ease, consensus sequences for splice sites in exon 3 were intact, but a 395C→G mutation was identified 24 bp upstream from the 3' splice site of exon 3. This mutation created a cryptic 5' splice site with a better consensus sequence for 5' splice sites than the natural 5' splice site of intron 3. A minor population of the IDS message was processed by using this cryptic splice site; however, no correctly spliced message was detected in leukocytes from this patient. The mutational topology of the IDS gene is presented.