Efficient Strategy for Molecular Diagnosis of Wilson Disease in the Sardinian Population

Abstract

Wilson disease (WD) is an autosomal recessive disorder of copper transport resulting from the defective function of a copper-transporting ATPase (ATP7B) (1)(2)(3). More than 200 disease-causing mutations have been identified (4). In the Sardinian population, WD has an incidence of ∼1 in 7000 live births (5). Using single-strand conformation polymorphism (SSCP) and sequencing methods for mutation analysis, we have characterized 92% of the chromosomes analyzed and identified 16 different WD-causing mutations, 6 of which (−441/−427del, 213–214delAT, 1512–1513insT, R778W, 2463delC, and V1146M) are relatively common and account for 85% of chromosomes (6). On the basis of these data, we developed a reverse dot-blot (RDB) method as a practical solution to mutation screening in this population. DNA samples from Sardinian WD patients carrying different combinations of the most common mutations (−441/−427del, 2463delC, V1146M, 213–214delAT, 1512–1513insT, and R778W) were used as controls. Our aim was to obtain the same PCR conditions for all six pairs of primers that were used to amplify the regions containing the six most common mutations. We therefore designed primers with an identical melting temperatures and tested their specificity first in single and then in multiplex PCRs. We also wanted to obtain relatively equal yields for all PCR products to obtain comparable signals using the RDB method. We therefore tested different concentrations for each pair of primers and finally established primer concentrations that allowed us to obtain approximately …