Screening strategies for a highly polymorphic gene: DHPLC analysis of the Fanconi anemia group A gene.
BACKGROUND
Patients with Fanconi anemia (Fanc) are at risk of developing leukemia. Mutations of the group A gene (FancA) are most common. A multitude of polymorphisms and mutations within the 43 exons of the gene are described. To examine the role of heterozygosity as a risk factor for malignancies, a partially automatized screening method to identify aberrations was needed. We report on our experience with DHPLC (WAVE (Transgenomic)).


METHODS
PCR amplification of all 43 exons from one individual was performed on one microtiter plate on a gradient thermocycler. DHPLC analysis conditions were 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 identified polymorphic PCR products or mutants.


CONCLUSIONS
We have defined the mutation screening conditions for all 43 exons of FancA using DHPLC. So far, 40 different sequence variations have been detected in more than 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 demand and costs. DHPLC is a valuable tool for reproducible recognition of known sequence aberrations and screening for unknown mutations in the highly polymorphic FancA gene.
Rischewski
J
J
Department of Paediatric Haematology and Oncology, University Childrens Hospital, Martinistr. 52, D-20246, Hamburg, Germany. rischewski@uke.uni-hamburg.de
Schneppenheim
R
R
D001483:Base Sequence; D002648:Child; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004252:DNA Mutational Analysis / Q000379:methods*; D017931:DNA Primers / Q000235:genetics; D005091:Exons; D005199:Fanconi Anemia / Q000150:complications / Q000235:genetics*; D005820:Genetic Testing / Q000379:methods*; D014644:Genetic Variation; D019337:Hematologic Neoplasms / Q000209:etiology / Q000235:genetics; D006579:Heterozygote; D006720:Homozygote; D006801:Humans; D015470:Leukemia, Myeloid, Acute / Q000209:etiology / Q000235:genetics; D009691:Nucleic Acid Denaturation; D016133:Polymerase Chain Reaction; D011110:Polymorphism, Genetic; D020641:Polymorphism, Single Nucleotide; D015203:Reproducibility of Results; D012307:Risk Factors
D017931:DNA Primers