ABSTRACT
BACKGROUND: Wilson's disease is an autosomal recessive disorder characterized by decreased biliary copper excretion and reduced copper incorporation into ceruloplasmin. The disease gene ATP7B maps to chromosome 13q14.3, contains 21 exons, and encodes a copper-transporting P-type ATPase. ATP7B mutations are scattered over the entire gene, and scanning methods to detect mutation carriers are in demand. We have tested the usefulness of denaturing high-performance liquid chromatography for mutation detection in Wilson's disease. METHODS: Genomic DNA from five Sardinian Wilson's disease families (32 individuals, 8 patients) was subjected to polymerase chain reactions for ATP7B exons 2-21 and the 5' untranslated region. PCR products were analyzed by chromatography and by direct sequencing. RESULTS: Three disease-causing mutations and seven sequence variants were detected by chromatography. Five patients were homozygotes for -441/-427del, and three were compound heterozygotes for V1146M plus 1512-13insT (N505X) and for -441/-427del plus V1146M, respectively. Eighteen unaffected individuals were mutation carriers. Sequence variants comprised V290V, A406S, L456V, R832K, A1140V, the novel K952R, and T991T. The novel intronic IVS18+6c>t change escaped detection by chromatography. CONCLUSIONS: Denaturing high-performance liquid chromatography is a dependable tool for ATP7B screening that is superior to traditional haplotyping. This method allows for fast, sensitive, and specific mutation detection and identification of carriers in Wilson's disease families.
