Clinical Chemistry 46: 1866-1867, 2000;
(Clinical Chemistry. 2000;46:1866-1867.)
© 2000 American Association for Clinical Chemistry, Inc.
Real-Time PCR Assay with Fluorescent Hybridization Probes for Rapid Genotyping of the CD14 Promotor Polymorphism
Michael Heesen1,a,
Dagmar Kunz2,
Rolf Rossaint1 and
Brunhilde Blömeke3
Departments of
1
Anesthesia,,
2
Clinical Chemistry and Pathobiochemistry,, and
3
Dermatology, University Hospital RWTH Aachen, Pauwelsstrasse 30, D-52074 Aachen, Germany
a Author for correspondence. Fax 49-241-8888406; e-mail mheesen{at}post.klinikum.rwth-aachen.de
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To the Editor:
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A soluble form of CD14 (1) activates endothelium and
smooth muscle (2). CD14 binds lipopolysaccharide,
the cell wall component of gram-negative bacteria. Upon
lipopolysaccharide binding, monocytes produce pro-inflammatory
cytokines and procoagulant activity. In view of the growing evidence
for a role of infection with gram-negative bacteria (3),
inflammation, and hypercoagulability in the onset of atherosclerosis,
two independent studies evaluated the frequency of a genetic
polymorphism within the promotor of the CD14 gene in
patients with myocardial infarction (MI) (4)(5). This
polymorphism consists of a single base exchange (C
T) at position
-260 (4) [corresponding to position -159 in study by
Unkelbach et al. (5)], with C introducing a
HaeIII restriction site.
The polymorphic site is located near the Sp 1 binding site of the
promotor (4). An increased risk for MI in patients
homozygous for the T allele was found
(4)(5). Moreover, Unkelbach et al.
(5) observed an even stronger association between the
TT genotype and the risk for MI in patients without
other risk factors such as smoking and hypertension. The odds ratio for
MI in normotensive nonsmoking TT homozygotes older than 62
years was 3.8 (5).
Because perioperative MI remains a major complication in surgical
patients (6), genotyping for the CD14 promotor polymorphism
could become a part of preoperative risk classification of surgical
patients.
The techniques reported for CD14 genotyping (restriction fragment
length polymorphism and single-strand conformation polymorphism
analysis) are time-consuming and require multiple manual steps. Because
a high throughput of samples is desirable for future studies, we
suggest a rapid-cycle PCR with fluorescently labeled oligonucleotide
hybridization probes on the LightCyclerTM
instrument (Roche Diagnostics) and subsequent fluorescent probe melting
point analysis, which allows genotyping within 60 min.
Genomic DNA samples from 100 healthy blood donors were extracted from
whole blood according to standard procedures. The reliability of the
proposed assay was confirmed by restriction enzyme digestion with
HaeIII.
PCR was performed in disposable capillaries (Roche Diagnostics) in a
reaction volume of 10 µL containing 1 µL of DNA (20–80 ng), 0.5
µmol/L each of the primers (sense,
5'-GGTGCCAACAGATGAGGTTCAC-3'; antisense,
5'-CTTCGGCTGCCTCTGACAGTT-3'), 1 µL of reaction buffer
[LightCycler DNA master hybridization probes 10x buffer (1x = 1.75
mmol/L); Roche Diagnostics], and 0.2 µmol/L each of the
probes. The detection probe specific for the T allele
(5'-TTCCTGTTACGGCCCCCCT-3') was labeled at the 3' end with
fluorescein. The anchor probe
(5'-GGAGACACAGAACCCTAGATGCCCTGCA-3') was labeled with
LightCycler Red 640 at the 5' end and modified at the 3' end by
phosphorylation to block extension. The PCR conditions were as follows:
initial denaturation at 95 °C for 120 s, followed by 60 cycles
of denaturation (95 °C for 0 s, 20 °C/s), annealing
(55 °C for 10 s), and extension (72 °C for 10 s). The
melting curve consisted of 1 cycle at 95 °C for 0 s, 45 °C
for 10 s, and then increasing the temperature to 95 °C at a
slope of 0.2 °C/s.
The fluorescence signal (F) was monitored continuously during the
temperature ramp and then plotted against the temperature
(T). These curves were transformed to derivative melting
curves [(-dF/dT) vs T].
Representative results for the three different genotypes
(TT, CT, and CC) are given in Fig. 1
. In the 100 patient samples, 27% were TT, 41% were
CT, and 32% were CC. The proposed technique and
the restriction enzyme technique gave identical results. The assay is
rapid and accurate and seems especially suited for routine laboratories
that process large numbers of samples.
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Figure 1. Representative derivative melting curves
[(-dF/dT) vs T] of the three CD14
genotypes.
· · · ·, TT; ——–, CT; - - -
-, CC.
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Acknowledgments
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We thank Olfert Landt (TIB MOLBIOL, Tempelhofer Weg 11-12, 10829
Berlin, Germany) for designing the hybridization probes.
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References
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Goyert SM, Ferrero E, Rettig WJ, Yenamandra AK, Obata F, LeBeau MM. The CD14 monocyte differentiation antigen maps to a region encoding growth factors and receptors. Science 1988;239:497-500.[Abstract/Free Full Text]
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Frey EA, Miller DS, Gullstein Jahr T, Sundan A, Bazil V, Espevik T, et al. Soluble CD14 participates in the response of cells to lipopolysaccharide. J Exp Med 1992;176:1665-1671.[Abstract/Free Full Text]
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Leinonen M, Linnanmäki E, Mattila K, Nieminen MS, Valtonen V, Leirisalo-Repo M, Saikku P. Circulating immune complexes containing chlamydial lipopolysaccharide in acute myocardial infarction. Microbiol Pathol 1990;9:67-73.
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Hubacek JA, Pit’ha J, Skodova Z, Stanek V, Poledne R. C(-260)T polymorphism in the promotor of the CD14 monocyte receptor gene as a risk factor for myocardial infarction. Circulation 1999;99:3218-3220.[Abstract/Free Full Text]
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Unkelbach K, Gardemann A, Kostrzewa M, Philipp M, Tillmanns H, Haberbosch W. A new promotor polymorphism in the gene of lipopolysaccharide receptor CD14 is associated with expired myocardial infarction in patients with low atherosclerotic risk profile. Arterioscler Thromb Vasc Biol 1999;19:932-938.[Abstract/Free Full Text]
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Lee TH. Reducing cardiac risk in noncardiac surgery. N Engl J Med 1999;341:1838-1840.[Free Full Text]
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