Genetics
A hidden CETP gene variant caused false positives in fluorescence-based TaqIB genotyping (Clin Chem 2001)
Original title: Fluorescence-based detection of the CETP TaqIB polymorphism: false positives with the TaqMan-based exonuclease assay attributable to a previously unknown gene variant
The CETP TaqIB polymorphism is linked to plasma CETP and HDL concentrations and to coronary artery disease progression, so this study tested two fluorescence-based genotyping methods against restriction fragment length polymorphism (RFLP) analysis in 150 coronary artery disease patients with known TaqIB genotype (50 each of B1/1, B1/2, and B2/2). TaqMan allele-specific PCR misclassified three B1/2 heterozygotes as homozygous B2. Melting curve analysis on the LightCycler flagged these same samples, plus four B1/B1 homozygotes, by an extra melting point at 59.1 degrees C, and DNA sequencing traced this to a previously unknown C270T substitution in intron 1, just nine base pairs from the TaqIB site, underscoring the need to validate new genotyping assays before routine use.
Original abstract
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.
Summary written by cetpinhibition.org from the published abstract; figures as published. Page updated 18 August 2026. Methods.