Allele dropout in PCR-based diagnosis of Wilson disease: Mechanisms and solutions

Background: We investigated the mechanisms leading to allele dropout-the nonamplification of 1 of the alleles-in PCR-based diagnosis of Wilson disease (WD). Methods: We extracted genomic DNA from blood samples from 6 WD patients (P1-P6) with allele dropouts detected in a previous study of WD in a Hong Kong Chinese population. We amplified the ATP7B gene by PCR and performed direct DNA sequencing of all exons of the ATP7B gene. To support the proposed mechanism of allele dropout, we used proofreading DNA polymerase, primer design avoiding single-nucleotide polymorphism sites, and duplex PCR. Results: Patients P1-P4 were all apparently homozygous for a known disease-causing mutation, c.2975C>T (p.P992L) in exon 13. Patient P5 was apparently homozygous for a novel mutation, c.2524G>A, and patient P6 was apparently homozygous for another known mutation, c.522_523insA (p.K175K-fs). In all cases, we determined that the patients were actually heterozygous for these mutations. Conclusion: Our results confirm that allele dropout is the mechanism causing apparent homozygosity of heterozygous mutations in these WD patients. (C) 2006 American Association for Clinical Chemistry.