Screening strategies for a highly polymorphic gene

TY - JOUR

T1 - Screening strategies for a highly polymorphic gene

T2 - DHPLC analysis of the Fanconi anemia group A gene

AU - Rischewski, J

AU - Schneppenheim, R

PY - 2001/1/30

Y1 - 2001/1/30

N2 - INTRODUCTION: 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.RESULTS AND DISCUSSION: 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.

AB - INTRODUCTION: 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.RESULTS AND DISCUSSION: 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.

KW - Base Sequence

KW - Child

KW - Chromatography, High Pressure Liquid

KW - DNA Mutational Analysis

KW - DNA Primers

KW - Exons

KW - Fanconi Anemia

KW - Genetic Testing

KW - Genetic Variation

KW - Hematologic Neoplasms

KW - Heterozygote

KW - Homozygote

KW - Humans

KW - Leukemia, Myeloid, Acute

KW - Nucleic Acid Denaturation

KW - Polymerase Chain Reaction

KW - Polymorphism, Genetic

KW - Polymorphism, Single Nucleotide

KW - Reproducibility of Results

KW - Risk Factors

M3 - SCORING: Journal article

C2 - 11179761

VL - 47

SP - 53

EP - 64

JO - J BIOCHEM BIOPH METH

JF - J BIOCHEM BIOPH METH

SN - 0165-022X

IS - 1-2

ER -