Screening strategies for a highly polymorphic gene: DHPLC analysis of the Fanconi anemia group A gene

Introduction: Patients with Fanconi anemia (Fanc) are at risk of developing leukemia. Mutations of the group A gene (FancA) are most common. A multitu de of polymorphisms and mutations within the 43 exons of the gene are descr ibed. To examine the role of heterozygosity as a risk factor fur malignanci es, a partially automatized screening method to identify aberrations was ne eded. We report on our experience with DHPLC (WAVE (Transgenomic)). Methods : PCR amplification of all 43 exons from one individual was performed on on e microtiter plate on a gradient thermocycler. DHPLC analysis conditions we re established via melting curves, prediction software, and test runs with aberrant samples. PCR products were analyzed twice native, and after adding a WT-PCR product. Retention patterns were compared with previously identif ied polymorphic PCR products or mutants. Results and discussion: We have de fined the mutation screening conditions for all 43 exons of FancA using DHP LC. So far, 40 different sequence variations have been detected in more tha n 100 individuals. The native analysis identifies heterozygous individuals, and the second run detects homozygous aberrations. Retention patterns are specific for the underlying sequence aberration, thus reducing sequencing d emand and costs. DHPLC is a valuable tool for reproducible recognition of k nown sequence aberrations and screening for unknown mutations in the highly polymorphic FancA gene. (C) 2001 Elsevier Science B.V. All rights reserved .