Rapid and reliable genotyping for the Toll-like receptor 4 A896G polymorphism using fluorescence-labeled hybridization probes in a real-time polymerase chain reaction assay.
BACKGROUND
The Toll-like receptor 4 (TLR4) is involved in immune response to endotoxin as well as the pathogenesis of atherosclerosis. The TLR4 gene was shown to carry a single-nucleotide polymorphism (A896G). We developed a rapid-cycle polymerase chain reaction (PCR) which allows for rapid genotyping and, therefore, may be useful in clinical risk assessment.


METHODS
Fluorescently labeled oligonucleotide hybridization probes were designed for the LightCycler instrument. A PCR product was generated in a single reaction followed by melting point analysis. Ninety-three German Caucasians were genotyped. The interleukin-1beta (IL-1beta) response to endotoxin was assessed after whole blood stimulation with endotoxin according to TLR4 genotypes.


RESULTS
The method suggested by us is a time-saving technique requiring no additional manual steps. Frequencies of the A and G allele were 0.95 and 0.05, respectively. The study population was in Hardy-Weinberg equilibrium. The specificity of the method was confirmed by sequencing. The IL-1beta levels were lower in carriers of the G allele.


CONCLUSIONS
This PCR assay is a rapid and reliable technique for TLR4 genotyping.
Heesen
Michael
M
Department of Anesthesia, Academic Medical Center, University of Amsterdam, Meibergdreef 9, 1100 DD Amsterdam, Netherlands. m.heesen@amc.uva.nl
Wessiepe
Martina
M
Kunz
Dagmar
D
Vasickova
Katarina
K
Blomeke
Brunhilde
B
D005838:Genotype; D006801:Humans; D006824:Hybridization, Genetic; D007375:Interleukin-1 / Q000097:blood / Q000235:genetics; D008562:Membrane Glycoproteins / Q000032:analysis / Q000235:genetics*; D015345:Oligonucleotide Probes; D016133:Polymerase Chain Reaction / Q000379:methods*; D011110:Polymorphism, Genetic; D011956:Receptors, Cell Surface / Q000032:analysis / Q000235:genetics*; D015203:Reproducibility of Results; D013696:Temperature; D051197:Toll-Like Receptor 4; D051193:Toll-Like Receptors
D007375:Interleukin-1; D008562:Membrane Glycoproteins; D015345:Oligonucleotide Probes; D011956:Receptors, Cell Surface; C495345:TLR4 protein, human; D051197:Toll-Like Receptor 4; D051193:Toll-Like Receptors