Determination of the frequencies of ten allelic variants of the Wilson disease gene (ATP7B), in pooled DNA samples

Wilson disease is an autosomal recessive disorder characterised by toxic ac cumulation of copper in liver, brain and other organs. The disorder is caus ed by mutations in the ATP7B gene, encoding a copper transporting P-type AT Pase. Based on the number of known patients with this diagnosis in Sweden, the prevalence can be estimated to 1 in 250 000 to 300 000, whereas the pre valence of Wilson disease has been estimated to be 1 in 30 000 in other pop ulations. We estimated the prevalence of Wilson disease by determining the Swedish population frequencies of two mutant alleles, making up approximate ly half the mutations in Swedish Wilson patients, in a large number of DNA samples. In addition we determined the allele frequencies of eight common s ingle-nucleotide polymorphisms (SNPs) in the ATP7B gene. For the analyses w e devised two strategies for analysing pooled DNA samples using the quantit ative minisequencing method. The two procedures allowed sensitive identific ation of rare mutant alleles present as a mixture with an excess of the nor mal allele, as well as accurate estimation of the frequencies of the common SNPs in a large pooled DNA sample.