Background: A number of polymerase chain reaction (PCR) based techniques like single-strand conformational polymorphism (SSCP), amplification refractory mutation system (ARMS)-PCR, semi-nested PCR and dinucleotide-repeat marker analysis have been used in the diagnosis of asymptomatic Wilson disease (WD) patients and the carrier status of WD families. In the present study, we explore the utility of mutation analysis in combination with restriction fragment length polymorphism (RFLP) in the genetic diagnosis of WD.
Aim: The study was planned to provide a molecular diagnostic tool for the diagnosis of asymptomatic WD patients as well as assessment of the carrier status of WD families.
Subjects and methods: Four WD families were analyzed in which parents and siblings showed no clinical manifestations or biochemical abnormalities. The parents of the WD patients were not consanguineous and had no family history of WD. Mutations in ATP7B were characterized using SSCP and DNA sequencing. Further, RFLP was developed for the analysis of characterized mutations in ATP7B from the WD patients, their parents and siblings.
Results: Three mutations, Q1256R, A1003T and I1102T, were characterized in WD patients, using SSCP and DNA sequencing. These mutations created/deleted restriction sites for AccII, Bsh1236I and EcoRI restriction enzymes respectively. Despite having no clinical manifestations nor any significant alteration in biochemical investigations, eight carriers and one asymptomatic WD patient were diagnosed in 13 members of the patients families by restriction digestion analysis.
Conclusion: The report demonstrates that mutation analysis in combination with RFLP is useful for diagnosis of asymptomatic WD patients as well as for the elucidation of the carrier status of the patients' family members. It is noteworthy that this combinational methodology provides a positive diagnosis in siblings/parents where biochemical parameters are ambiguous.