ATP7B Mutation Analysis In Wilson Disease Patients With Liver Injuries

Wilson disease is an autosomal recessive disease caused by mutation of ATP7B gene, the mutated gene results in a defect of copper excretion, the overload copper mainly toxic accumulate in the liver, brain and corneas and then results in injures of these organ or tissues. The worldwide prevalence of Wilson disease is estimated to be between 1 in 30,000 and 1 in 100,000, and it is seen more frequently in China. There is a obvious locality difference in ATP7B mutation, almost all the country have its own hot spot mutation. For the white people of European and American, the most common mutation is p.H1069Q which is situated in exon 14, the frequency of this mutation is between 20%- 70%. In China, the hot spot mutation is p.R778L which is situated in exon 8, the frequency of this mutation is between 11.4%-60%. Up to now, most research about chinese Wilson desease patients only study exons which have hot spot mutations, a small part of research do the sequencing of all exons, and also much less research do the whole family members sequencing study. Since the ATP7B gene was cloned by Bulls et al in 1993, a lot of data about the mutational analysis of ATP7B and genotype-phenotype correlation of Wilson disease was published, to date, almost 400 variations reported as causative of WD, but the whole pathologic mechanism of this old diasese is still unclear, as the patients who share the same mutations show the exactly different phenotype, and about 5% Wilson disease patients only have one or non-disease causing mutation.In this work, a total of 17 Wilson disease patients from northeast China are included, 12 of them are male, the other 5 are female. All of them are Han Chinese, four of them are individual patients, the other thirteen patients come from twelve family, and forty family members of those families participate in this study. 4 cc of peripheral blood was drawn from each patient by EDTA tube, and DNA was extracted from these samples by the classic phenol- chloroform method. DNA samples were amplified by PCR using 26 primers corresponding to all 21 exons, and they were subsequently sequenced. After all the exons of the index patients were directly done sequencing, only the exons of patients family members which had found mutations in index patients were done the sequencing. Using direct sequencing methods, 21 different mutations were detected, 10 of them were disease-causing varients, include 9 missense mutation (p.V659E, p.S744F, p.R778L, p.A874V, p.T888P, p.R919G, p.S975Y, p.P992L, p.N1270S) and one splice site mutation (IVS18-1 del G). The other 11 mutations are SNPs, 5 of them are missense mutation (p.A406S, p.L456V, p.K832R, p.R952K, p.A1140V), 2 of them are nonsense mutation (p.S88S, p.L770L), the other 4 mutations are in intron (IVS1-118ins CGCCG, IVS1-74A>C, IVS10-25A>G, IVS18+6 T>C). Thirteen out of 17 patients had two disease-causing mutations; another three had only one mutation found, one patient had no mutation identified. Also, we have identified three new mutations (p.V659E, p.S744F, IVS18-1delG) and two new polymorphisms (IVS1-118ins CGCCG, p.S88S). Using the directly sequencing mehtod, we identified two siblings as Wilson disease patients who were asymptomatic and 28 Wilson desease gene carriers from 12 Chinese Wilson disease pedigrees. Something interesting in this study is that twins with the same genetype and live in the same environment but show up total different phonetype. The prohand patient had both neurological and hepatic symptoms, but his brother has no signs and symptoms. In this cohort, p.R778L was the most common mutation and was identified in 12 WD patients (70.5%), two of them were homozygous of this mutation. In this study, the data suggest that there might be a correlation between p.R778L mutation and liver impairment. An p.R778L homozygote is also at greater risk for early-onset hepatic symptoms. The patients with multiple mutations which suspected non-disease causing, looks like a slowly development pathogenetic condition and lightly level of clinical course compare with patients who only have two disease causing mutations. Wilson disease is an autosomal recessive disease, so, though the direct sequencing of ATP7B gene, we can found the disease causing mutations, then we can screen for prohand patient's family and find out the carriers and asymptomatic patients. The fact that affected patients within the same family who have the same mutation can have very different phenotypes suggests that there are other factors impacting the presentation of the autosomal recessive disorder.In this study, though the direct sequencing of ATP7B gene, we can found the hot spot Wilson disease causing mutations, and then base on the clinical feature of the patients to find out the genotype-phenotype correlation. And also, though the whole family screen and ATP7B gene analysis, we can try to analyze other factors impacting the presentation of the autosomal recessive disorder to lay foundations for the further work of Wilson disease.