Morbus Wilson: Identification of three additional novel mutations in the ATPase7B gene

Abstract

Background: Mutations in the copper transporting P-type ATPase gene have been reported to result in the clinical phenotype Wilson disease. The disease is a rare inherited autosomal recessive disorder, characterized by impaired incorporation of copper into ceruloplasmin and by impaired excretion via the bile. The gene consists of 21 exons and encodes a protein of 1,465 amino acids. About 190 mutations have been detected throughout the whole gene sequence with certain accumulation in hot spots. The underlying mutations are very heterogeneous, thus the fast and reliable genetic screening for mutations as the underlying cause of the disease is hampered. The aim of this investigation was to assess novel mutations in the recently discovered Wilson disease gene. Methods: We screened 57 individuals of european origin for mutations in the 21 exons and their flanking intronic sequences using direct sequence analysis. All exons and exon/intron boundaries of patients suffering from Wilson disease were amplified by PCR. Subsequently, cycle sequencing of the purified PCR products using the fluorescent dye terminating system (PE Applied Biosystems) was performed. Detected mutations were confirmed using single strand conformation analysis of the PCR products. Results: So far we detected in 114 analyzed alleles 22 disease causing mutations affecting the ATPase 7B gene. Ten of these mutations were novel mutations (4 missense, 2 nonsense, 1 insertion, 2 deletions, t splice site) (Hum Murat 14 (1):88; 18(3):278). The mutations Pro76OLeu, Leu1305Pro und GIn1351Stop in the exons 8, 19, and 20 have not yet been reported. All patients were heterozygous for the mutation. The most common HislO69GIn mutation was found in 30 of 57 (53%) patients. 14 patients were homozygous carriers (47%), 16 patients were heterozygous carriers. The His1069GIn occurred with an allele frequency of 38.5% in this cohort. Conclusions: These data confirm, that Wilson disease in our patients results from a frequent mutation His1069GIn and/or from a large number of rare mutations. The mutation heterogeneity complicates genetic testing to confirm the diagnosis of WD, although DNAbased genetic analysis is still an important supplement to clinical diagnostics. The most recommendable procedure for genetic analysis is the analysis of the common mutation His1069GIn using PCR and subsequent restriction endonuclease digestion. In the absence of homozygosity of this mutation direct analysis of all exons including the promoter is required.