Fluorescence-based detection of the CETP TaqIB polymorphism: false positives with the TaqMan-based exonuclease assay attributable to a previously unknown gene variant.
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.
Teupser
D
D
Institute of Laboratory Medicine, Clinical Chemistry, and Molecular Diagnostics, University Hospital Leipzig, Liebigstrasse 27, 04103 Leipzig, Germany. daniel@teupser.de
Rupprecht
W
W
Lohse
P
P
Thiery
J
J
D002352:Carrier Proteins / Q000235:genetics*; D053480:Cholesterol Ester Transfer Proteins; D002788:Cholesterol Esters / Q000378:metabolism*; D003327:Coronary Disease / Q000235:genetics; D015252:Deoxyribonucleases, Type II Site-Specific; D005189:False Positive Reactions; D005838:Genotype; D006023:Glycoproteins; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods; D011110:Polymorphism, Genetic; D012150:Polymorphism, Restriction Fragment Length; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA
C506642:CETP protein, human; D002352:Carrier Proteins; D053480:Cholesterol Ester Transfer Proteins; D002788:Cholesterol Esters; D006023:Glycoproteins; D015252:Deoxyribonucleases, Type II Site-Specific; C027046:TCGA-specific type II deoxyribonucleases