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Published on: April 13, 2012
Fluorescence-based detection of the CETP TaqIB polymorphism: false positives with the TaqMan-based exonuclease assay
D Teupser1, W Rupprecht, P Lohse
1Institute of Laboratory Medicine, Clinical Chemistry, and Molecular Diagnostics, University Hospital Leipzig, Liebigstrasse 27, 04103 Leipzig, Germany. daniel@teupser.de
Insights
A new C270T polymorphism in the cholesteryl ester transfer protein (CETP) gene caused misclassification of TaqIB genotypes using the TaqMan system. Melting curve analysis detected this novel polymorphism, highlighting the need for thorough evaluation of new gene analysis methods.
Area of Science:
- Genetics and Molecular Biology
- Cardiovascular Disease Research
Background:
- The TaqIB polymorphism in the cholesteryl ester transfer protein (CETP) gene is linked to HDL levels and coronary artery disease (CAD) progression.
- Accurate genotyping is crucial for understanding genetic predispositions to CAD.
Purpose of the Study:
- To evaluate the performance of two novel fluorescence-based detection systems for TaqIB genotyping.
- To identify potential discrepancies in genotype determination between new and established methods.
Main Methods:
- Analysis of TaqIB genotypes in 150 CAD patients using restriction fragment length polymorphism (RFLP) as a reference.
- Comparison with fluorescence-based TaqMan PCR and melting curve analysis (LightCycler).
- Confirmation of findings using DNA sequencing.
Main Results:
- TaqMan system misclassified three heterozygous B1/2 patients as homozygous B2.
- Melting curve analysis revealed an additional melting point in misclassified samples and some B1 homozygotes.
- DNA sequencing identified a novel C270T polymorphism near the TaqIB site in the CETP gene.
Conclusions:
- A previously unknown C270T polymorphism in the CETP gene caused TaqMan system misclassifications of TaqIB genotypes.
- LightCycler's melting curve analysis successfully detected the C270T polymorphism.
- New gene analysis systems require rigorous validation before routine clinical application.
Background:
Previous studies have shown an association between the TaqIB polymorphism of the cholesteryl ester transfer protein (CETP) gene with plasma CETP and HDL concentrations and the progression of coronary artery disease (CAD). The aim of the present study was to determine the performance of two new fluorescence-based detection systems in the analysis of the TaqIB genotype.
Methods:
CAD patients (n = 150) with known TaqIB genotype, as determined by restriction fragment length polymorphism (RFLP) analysis, were selected, including three groups of 50 patients, carrying the B1/1, B1/2, and B2/2 genotypes, respectively. The genotypes were also analyzed by fluorescence-based allele-specific TaqMan PCR and melting curve analysis (LightCycler). In addition, DNA sequencing was applied.
Results:
The TaqIB genotypes obtained by fluorescence analysis corresponded to those determined by RFLP analysis with the exception of three heterozygous patients (B1/2), who were misclassified as homozygous B2 carriers with the TaqMan system. Melting curve analysis of these samples demonstrated an additional melting point at 59.1 degrees C, which was also found in four patients homozygous for the B1 allele. DNA sequencing revealed a previously unknown C270T nucleotide exchange in intron 1 of the CETP gene, only nine base pairs from the TaqIB site.
Conclusions:
Determination of the TaqIB polymorphism with the TaqMan system led to misclassifications because of a previously unknown C270T polymorphism of the CETP gene. The base substitution was detected with the LightCycler because of the occurrence of an additional melting point. Our data indicate the importance of thorough evaluation of new gene analysis systems before using them on a routine basis.
