| Background. Wilson disease (MIM ;Methods. Seventy-six Wilson disease patients from 65 unrelated Hong Kong Chinese families were recruited. The ATP7B gene was analyzed by PCR, sequencing and haplotype analysis. Primers were specially designed to avoid allele dropout. The p.R778L/p.L770L status in 660 normal subjects was assessed to estimate the local prevalence. Allele age of p.R778L/p.L770L was determined by the smallest homozygosity region between D13S301 and D13 S270. Receiver operating characteristic curve analysis of serum ceruloplasmin was performed among 168 ATP7B genotyped subjects. Finally, we developed real-time amplification refractory mutation system PCR to detect 36 ATP7B mutations using SYBR green fluorescent dye.;Results. We improved the molecular analytical accuracy by correcting 17 errors observed in previously published primers and we designed a new set of PCR primers that avoid allele dropout. We characterized 42 mutations of ATP7B gene showing a Hong Kong Chinese specific pattern. Seventeen were novel. The three commonest mutations were p.R778L (17.3%), p.P992L (13.4%) and p.I1148T (8.7%). There was no genotype and phenotype correlation. Local prevalence of Wilson disease was estimated to be 1 in 5,400. We also developed a one-step, accurate, reproducible and cost-efficient real-time amplification refractory mutation system PCR detecting 36 ATP7B mutations in only three hours. We have established the receiver operating characteristic curve of serum ceruloplasmin concentration from 193 genotypically confirmed subjects (57 WD patients, 71 their family members, 25 patients of validation group and 40 subjects from the reference range group). Level less than 0.14g/L rendered the best positive and negative predictive values of 100% and 97.1% respectively.;Conclusions. This is the first mutational study of Wilson disease in Hong Kong Chinese. The mutation detection rate was 97.6% that was the highest among Asian studies. The genetic heterogeneity is the most diverse. We established a half-gene screening approach (exons 2, 3, 8, 10, 11, 12, 13, 16, 18 and 19) that can achieve 90% mutation coverage. Wilson disease is not rare in Hong Kong Chinese and there is a need for the consideration of population screening. Based on the perfect linkage disequilibrium of p.R778L/p.L770L, we postulated that the present population inheriting this founder mutation, residing in Hong Kong, Mainland China, Taiwan, Japan and Korea, have descended from the same ancestor aged 5,500 years. |
