Mutational studies in Korean patients with Wilson disease and the expression and transcriptional regulation of the ATP7B gene

Abstract

Wilson disease (WD) is an autosomal recessive disorder of copper transport. Its general characteristics are defective biliary excretion of copper and impairment in the incorporation of copper in ceruloplasmin. The product of the WD gene is a copper-transporting P-type ATPase (ATP7B). The ATP7B gene has 21 exons and encodes a protein of 1,465 amino acids. WD occurs once in 30,000-100,000 in most populations, but its exact incidence has not yet been determined in Korea. To identify mutations in Korean patients with WD, single-strand conformation polymorphism (SSCP) analysis followed by DNA sequencing of PCR amplified exons was performed. Of the 12 sequence changes identified in the ATP7B gene, three mutations (A874V, L1083F, and 2304delC) were novel causing WD. Arg778Leu, the most frequently reported mutation of this enzyme, was found in one of three unrelated patients studied. The novel single nucleotide deletion, 2304delC, was found in one patient. Since a mutation at cDNA nucleotide 2302 (2302insC) had been previously described, this region of the ATP7B gene may be susceptible to gene rearrangements causing Wilson disease. Among these WD patients, six new sequence polymorphisms (3244-5C→T, 1948-4C→T, 1870-2delA, Val864Ile, Val1109Met, and Leu770Leu) and two previously described (Lys832Arg and Val1140Ala) were also identified. To investigate the function of the ATP7B protein, the 4.5 kb full-length cDNA of the ATP7B gene encoding a protein of 1,465 amino acids was cloned. During the cloning of the ATP7B gene, the exon 6-7 and 6-7-8 deletion forms were detected in a human adult liver cDNA and fetal liver library, respectively. The each form was designated A△6-7 and F△6-8. Moreover, five alternative-spliced forms were also found by RT-PCR in HepG2 cells. The exon 8, exon 5-6-7, and exon 5-6-7-8 deletion forms were named H△8, H△5-7, and H△5-8, respectively. Polyclonal antibody was generated against the copper-binding domain of ATP7B, which was overexpressed and pu...