METHODS Patients MPS IVA patients were diagnosed at each collaborating institute and the Department of Pediatrics, Saint Louis University (St. Louis, MO). Twenty-eight MPS IVA patients were analysed. GALNS deficiency was demonstrated in all cases by enzyme assay. Plasma and urine KS concentrations were measured as described previously, and the reagents were obtained from Seikagaku (Tokyo, Japan). 16 The subjects studied included 16 Americans, four Canadians, two Austrians, two Pakistanis, two Japanese, one Mexican, and one New Zealander. Clinical data and the racial background of each patient are reported in table 1. Peripheral blood samples were collected from the patients after obtaining an informed consent at each collaborating institute.

Mutation analysis of GALNS gene Genomic DNA was extracted by a standard method. Five sets of primers covering all 14 exons and their exon–intron boundaries were assigned to produce five amplicons (0.3–5.8 kb in length). In each amplicon (F1–F5), 200 ng of genomic DNA were mixed with a set of primers in a total volume of 50 ml, including dNTPs, DTT, dimethyl sulfoxide (DMSO), and Taq polymerase. Except for F1 (0.3 kb) for exon 1, PCR fragments included multiple exons, amplified by a long PCR amplification method according to the manufacturer’s instructions (Gibco BRL, Rockville, MD) with the addition of 2.5–5% of DMSO or 1 ml Perfect Match (Stratagene, La Jolla, CA). All PCR amplification reactions were performed on a Perkin-Elmer 9700 Thermal Cycler. The PCR products were directly sequenced using fluorescent-labelled dideoxynucleotides (ABI, Foster City, WA). The positions of PCR primers and the sizes of PCR product are shown in fig 1 together with the picture of the