Carsten A. Buettner

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

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.

Inhibition of transthyretin-met30 expression with inosine15.1-hammerhead ribozymes in cell culture

Hereditary amyloidosis is primarily caused by mutations within the transthyretin gene. More than 75 mutations within transthyretin have been reported in causing amyloidosis. The most common mutation is the val30met mutation in the transthyretin protein (TTR-met30) caused by a mononucleic substitution from G to A (GUC to AUC) in the transthyretin gene resulting in the exchange for the amino acids valine to methionine in the corresponding protein sequence. The aim of this work is the development of a specific cleavage of TTR-met30 mRNA in the cell culture system using hammerhead ribozymes. We showed previously that chemically modified nuclease stable Inosine(15.1)-Hammerhead ribozymes are able to target the TTR-met30 mRNA with high specificity on the RNA level (Biochem. Biophys. Res. Commun. 260, 313-317, 1999). Now we present data confirming our observations on the cellular level. We used the wild-type human normal (hn) TTR expressing cell line HepG2 and the stable transfected cell line 293-TTR-met30 for TTR-met30 experiments. We cleaved the TTR-met30 and hnTTR mRNA with specific nuclease stable chemically modified Inosine(15.1)-Hammerhead ribozymes and analyzed the protein after immunoprecipitation and subsequent Western blotting. We were able to downregulate the TTR concentration by 54.5% (100% = 1.5 mg/l TTR) and also specifically to target the TTR-met30 expression in the cell culture system. The therapeutic effect was improved using cationic liposomes resulting in a total downregulation by 92.1 and 62.7% targeting hnTTR mRNA and TTR-met30 mRNA, respectively. The successful employment of Inosine(15.1)-Hammerhead ribozymes in cell culture is therefore a promising tool for the development of a gene therapeutic strategy for hereditary amyloidosis.