Non-radioactive detection of five common microsatellite markers for ATP7B gene in Wilson Disease patients.

Haplotype analysis using microsatellite markers is a useful indicator of specific mutations and is often exploited as the first large-scale screening technique to carry out the molecular characterization of the disease gene in probands from a specific population. However, the methodologies available are still cumbersome and require the use of either radioactive compounds or specialized equipment suitable to follow fluorescent dyes. Both these techniques may not be available for newly-developing clinical laboratories. We have set up a sensitive and easy-to-use protocol to characterize five closely spaced, highly polymorphic microsatellite polymorphisms (CA repeats) that span the Wilson Disease (WD) region i.e., D13S316, D13S133, D13S301, D13S314, D13S315. The technique described here for the analysis of the WD gene microsatellite system relies on the quick detection method of silver staining, avoiding the use of toxic or sophisticated equipment. This approach could be the method of choice to implement molecular genetic testing in clinical laboratories, even those not especially equipped for DNA analysis and in particular in newly-developed molecular genetics centers in countries whose population has not yet been characterized for WD-causing ATP7B gene mutations.