Genetic Analysis Of The Wilson Disease Pedigrees

Objective:Wilson disease (WD) is a rare autosomal recessive disorder of hepatic copper metabolism leading to copper toxic accumulation in hepatocytes and in extrahepatic organs such as the brain and the corner. The world prevalence is about1in30,000, with a carrier rate1in90. Its main clinical manifestations including hepatic disease, neurological symptpms, psychiatric disturbances and detection of Kayser-Fleischer rings. ATP7B gene (MIM#277900) is the only gene identified associate with WD, encodes a copper transporting ATPase, ATP7B. Mutation of ATP7B gene would cause loss of ATP7B function, it’s the basis for reduced hepatic biliary copper excretion and reduced incorporation of copper into ceruloplasmin. The prognosis of most WD patients by decoppering chelation is excellent, and the earlier the treatment, the better the prognosis. Due to the age of onset and progress varies, organ damage differences, leading to the initial symptoms of WD varied and clinical manifestations are complex, resulting in the clinical diagnosis difficult. If left untreated, WD will processing become a potentially lethat disorder. So, timely and accurately diagnose WD is very important.Purpose:In this research, we aimed at identifying all the WD pedigrees with clinical diagnosis or clinical suspicion by molecular biological techniques, helping patients with timely treatment and performing prenatal genetic diagnosis for their parents’current pregnancy or re-pregnancy.Methods:1. All the WD patients with clinical diagnosis or clinical suspicion were detected all coding regions and the intron/exon boundaries of ATP7B depending on specific PCR sequencing analysis. RT-PCR was performed to investigate if the new splicesite mutation affected the gene splicing site. In order to exclude the possibility that the detected gene variation might be a polymorphism, DNA samples of100healthy control individuals were investigated by direct sequencing in the same position.2. Patients with only one mutation or no mutations being found, would furtherly detect the promoter region of ATP7B gene by directly sequencing.3. Patients without mutations being found both in coding-region and promoter region, will be confirm whether the ATP7B exon exist deletions or duplications depending on Multiplex Ligation dependent Probe Amplification (MLPA).4. Using directly sequencing and linkage analysis for prenatal diagnosis of a pregnant women, while the couples were WD carriers.Results:1. In this study,14Chinese pedigrees affected with Wilson disease were analyzed, identified homozygous mutations or compound heterozygou mutations of ATP7B gene in12patients. The other two patients only found one mutation. Detection rate was92.9%. A total17different mutations were identified.2. There was one polymorphism at positions-190(transcription start site as+1) of the promoter region of ATP7B gene identified. Normal controls and WD patients all showed the polymorphism.3. Prenatal diagnosis for three cases of WD high-risk fetuses, fetus of family11was detected heterozygou mutation c.2975C>T of ATP7B gene, fetus of family9was found compound heterozygous mation c.2383C>T and c.4005delG of ATP7B gene, fetus of family14was idetified no mutations in ATP7B gene. Conclusion:1. Totally6novel mutations of ATP7B gene were found, expanding the mutational spectrum of ATP7B.2. One polymorphism at position-190(transcription start site as+1) of the promoter region of ATP7B gene was identified.3. Combining directly sequencing and MLPA is able to provide fast and accurately prental diagnosis for WD pedigrees.