Detection of four novel mutations in the iduronate-2-sulphatase gene by single-strand conformation polymorphism analysis of genomic amplicons.

Abstract

Hunter syndrome (mucopolysaccharidosis type II, MPS II; McKusick 309900) is caused by the deficiency of iduronate-2-sulphatase (IDS, EC 3.1.6.13) enzyme activity (Bach et al 1973). The X-linked IDS gene comprises nine exons and eight introns and encodes a polypeptide of 550 amino acids (Wilson et al 1993). About 50 small mutations in the IDS gene have been detected by an approach using reverse transcription-linked polymerase chain reaction (RT-PCR) (Jonsson et al 1995). In the present investigation, an alternative approach which combined exon-by-exon PCR amplification of the IDS genomic sequence with single-strand conformation polymorphism (SSCP) analysis was developed for screening mutations in the IDS gene. Five unrelated patients, designated patients 1-5, with the severe type of Hunter syndrome were investigated. All five patients were diagnosed by the demonstration of the IDS enzyme deficiency in serum or cultured fibroblasts. For mutation analysis, nine pairs of IDS exon-specific primers, designated ElL, R; E2L, R; E3L, R; E4L, R; E5L, R; E6L, R; E7L, R; ESL, R; and E9L, R, were designed from the IDS genomic 5' and 3' non-coding regions and exon/intron boundary sequences (Wilson et al 1993). The synthesized primer pairs were used in a standard PCR to generate IDS exon I to IX specific amplicons with size ranging from 129bp to 595bp. The IDS exon-specific amplicons were subjected to SSCP analysis in order to identify an alteration in mobility by MDE gel electrophoresis (AT Biochem). The amplicons with altered mobility were directly sequenced using a ATaq cycle sequencing kit (United States Biochemical). Two unique missense mutations in exon VII were identified: D334G (GAT to GGT, At°°I--~G) in patient 3 and Gl°°6--~A (TGgt to TAgt; italic denotes intron sequence) in patient 4. A previously reported mutation, $333L (TCG to TTG, C99s--~T in exon VII), was found in patient 2. Two novel nonsense mutations were defined: Y234X (TAC to TAA, C7°2-~A in exon V) in patient 1 and Q465X (CAG to TAG, C1393---)T in exon IX) in patient 5. The GI°°6~A mutation eliminates a NlaIII recognition site and the Q465X mutation introduces an AluI recognition site, which allow a convenient PCR-based restriction enzyme assay for carrier detection. The $333L mutation was defined as one of the several CpG dinucleotide 'hot spots' in the IDS gene (Jonsson et al 1995). The identification of four novel mutations in the IDS gene provides further evidence of mutational heterogeneity in Hunter syndrome.