RESUMO
AIMS: Wilson disease (WD) is an autosomal recessive disorder of copper metabolism, characterized by its accumulation in tissues which results in hepatic, neurological, and/or psychiatric symptoms. The aim of this study was to investigate the genetics of WD in Croatian patients. METHODS: Correlation of the clinical presentation subtype and the age at onset of the diagnosis of WD with the ATP7B genotype was investigated in a group of Croatian WD patients. DNA from peripheral blood samples was tested for the p.His1069Gln by direct mutational analysis and other polymorphisms were identified by sequence analysis of coding and flanking intronic regions of ATP7B gene. RESULTS: In the group of 75 WD patients of Croatian origin, 18 different mutations in ATP7B gene were detected, three of which were novel. The p.His1069Gln mutation was most frequent, being detected in 44 Croatian WD patients (58.7%). Most ATP7B mutations (90.4%) were located in exons 5, 8, 13, 14, and 15. CONCLUSIONS: Clinical diagnosis of WD was confirmed in 59 patients by detecting mutations on both ATP7B alleles. The age at onset of WD and the type of WD clinical presentation showed no significant correlation with the ATP7B genotype.
Assuntos
Adenosina Trifosfatases/genética , Proteínas de Transporte de Cátions/genética , Degeneração Hepatolenticular/genética , Adenosina Trifosfatases/sangue , Adulto , Alelos , Proteínas de Transporte de Cátions/sangue , ATPases Transportadoras de Cobre , Croácia , Análise Mutacional de DNA , Éxons , Feminino , Estudos de Associação Genética , Degeneração Hepatolenticular/enzimologia , Humanos , Masculino , Pessoa de Meia-Idade , Mutação , Polimorfismo Genético , Polimorfismo de Fragmento de RestriçãoRESUMO
Approximately 520 Wilson disease-causing mutations in the ATP7B gene have been described to date. In this study we report DNA and RNA analyses carried out for molecular characterization of a consensus sequence splicing mutation found in homozygosity in a Swiss Wilson disease patient. RNA analysis of 1946 +6 TâC in both the peripheral lymphoblasts and liver resulted in the production in the propositus of only an alternative transcript lacking exons 6, 7, and 8 resulting most likely in alterations of cell biochemistry and disease. The patient presents an early form of severe hepatic disease characterized by hepatosplenomegaly, reduced hepatic function, anemia and thrombocytopenia indicating that 1946 +6 TâC is a severe mutation. Since identical results were obtained from both peripheral lymphoblasts and liver they also suggest that RNA studies of illegitimate transcripts can be safely used for molecular characterization of ATP7B splicing mutations, thus improving genetic counseling and diagnosis of Wilson disease. Moreover these studies, contribute to reveal the exact molecular mechanisms producing Wilson disease.
Assuntos
Adenosina Trifosfatases/genética , Proteínas de Transporte de Cátions/genética , Degeneração Hepatolenticular/diagnóstico , Sequência de Bases , Criança , Sequência Consenso , ATPases Transportadoras de Cobre , Feminino , Degeneração Hepatolenticular/genética , Homozigoto , Humanos , Técnicas de Diagnóstico Molecular , Mutação Puntual , Isoformas de Proteínas/genética , Sítios de Splice de RNA/genética , Análise de Sequência de RNA , Transcrição GênicaRESUMO
Wilson disease is an autosomal recessive disorder caused by defective function of the copper transporting protein ATP7B. Approximately 520 Wilson disease-causing mutations have been described to date. In this study we report the use of DNA and RNA analysis for molecular characterization of a gross deletion of the ATP7B gene detected in homozygosity in a Wilson disease patient. The c.51+384_1708-953del mutation spans an 8798 bp region of the ATP7B gene from exon 2 to intron 4. The results obtained suggest that the combination of DNA and RNA analyses can be used for molecular characterization of gross ATP7B deletions, thus improving genetic counselling and diagnosis of Wilson disease. Moreover these studies, help to better establish the molecular mechanisms producing Wilson disease.
Assuntos
Adenosina Trifosfatases/genética , Proteínas de Transporte de Cátions/genética , DNA/análise , Degeneração Hepatolenticular/genética , RNA/análise , Análise de Sequência de DNA/métodos , Análise de Sequência de RNA/métodos , Adolescente , Sequência de Bases , Consanguinidade , ATPases Transportadoras de Cobre , DNA/química , Éxons , Genes Recessivos , Aconselhamento Genético , Homozigoto , Humanos , Íntrons , Itália , Masculino , Dados de Sequência Molecular , Reação em Cadeia da Polimerase Multiplex , RNA/química , Deleção de SequênciaRESUMO
Wilson's disease (WD) is an autosomal recessive disorder caused by a defective function of the copper transporting ATP7B protein. Analysis of ATP7B gene in the Sardinian population revealed the presence of six common mutations that together account for 85% of WD chromosomes. We have developed an automated approach for the detection of these 6 common Sardinian mutations based on TaqMan technology. Ten DNA samples of WD patients carrying different combinations of the six most common Sardinian mutations and normal controls previously analysed were used in triplicate to set up the allelic discrimination assays. The system was validated in 96 samples obtained from WD patients carrying different combinations of the most common mutations under investigation. The results showed that allelic discrimination is a valid method that could be used for efficient diagnosis of single cases but also for a mass screening.
