Mucopolysaccharidosis IVA: screening and identification of mutations of the N-acetylgalactosamine-6-sulfate sulfatase gene.

Mutations causing mucopolysaccharidosis IVA in 15 Japanese and one Caucasian patient were characterized. To screen these mutations, we used a combination of single strand conformation polymorphism analysis and heteroduplex analysis for PCR products of targeted cDNA or genomic DNA. Various small mutations were identified in 23 of 26 alleles, while the other six alleles had large rearrangements. Cycle sequencing of PCR products revealed 15 different mutations, including 12 missense, one nonsense, one frame shift (2 bp deletion) and one splice site mutation, in accord with the broad range of clinical phenotypes. Two alleles have different mutations in the same nucleotide position of exon 3 (R94C, CGC-->TGC; R94G, CGC-->GGC), diagnosed by sequencing and by allelic-specific oligohybridization (ASO). One allele had two amino acid changes, E450V in exon 12 and V488M in exon 13, thereby indicating a double point mutation. All 16 mutations reported were confirmed by restriction enzyme assay or by allelic-specific oligohybridization. Transfection of mutagenized cDNAs into patients' fibroblasts showed that all mutations caused completely deficient or markedly decreased N-acetylgalactosamine-6-sulfate sulfatase (GALNS) activity, thereby indicating that these mutations were responsible for the enzyme deficiency.