Linkage Analysis based on Four Microsatellite Markers to Screen for Unknown Mutation in Families with Wilson Disease

Wilson disease (WD) is a rare autosomal recessive disorder of copper metabolism due to mutations in ATP7B gene on the chromosome 13. Linkage analysis using microsatellite markers is a powerful screening technique to identify mutant chromosomes especially in affected families with unknown mutations. Previous studies in southern Iran have failed to identify mutations in the ATP7B in some clinically diagnosed cases. Hence, the present study was undertaken to provide a screening method for these WD affected families.

Genomic DNA was prepared from the peripheral blood of 12 WD affected families. Four short tandem repeat (STR) markers around the WD locus (D13S301, D13S296, D13S201, and D13S297) were amplified by multiplex polymerase chain reaction (PCR) and amplified DNA fragments were analyzed by polyacrylamide gel electrophoresis (PAGE). In addition, 24 samples were amplified by PCR using fluorescent labeled primer and then examined by capillary electrophoresis (CE). Finally, haplotype and genotype analysis was done for each family.

According to the results of linkage analysis by STR markers, the genotypes of all the children related to the WD families were predicted. However, the genotypes of 8 persons remained unclear due to uninformative markers. This investigation indicated that 3 out of 4 selected STR markers were informative among Iranian population.

Based on the results, linkage analysis by STR markers is a powerful method for detection of potential carriers and presymptomatic WD homozygotes in families with at least one previously affected child. This approach is an efficient, accurate, and cost-effective method that can be used in clinical laboratories, especially in recently developed molecular genetics centers in countries whose patients have not yet been diagnosed for WD-causing ATP7B gene mutations.