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Real-time PCR assay with fluorescent hybridization probes for rapid interleukin-6 promoter (-174G [right arrow] C) genotyping.

To the Editor:

Interleukin-6 (IL-6) is a central protein in the regulation of the inflammatory and immunologic response (1). A G [right arrow] C polymorphism at position -174 has been associated with osteoporosis (2), juvenile rheumatoid arthritis (3), and atherosclerosis (4) with increased risks in the absence of the CC genotype.

[FIGURE 1 OMITTED]

Restriction fragment length polymorphism analysis has been used for IL-6 genotyping, but it is time-consuming and requires multiple manual steps. To improve throughput, we developed a rapid-cycle PCR method on the LightCycler [TM] instrument (Roche Diagnostics) with fluorescent probe melting analysis. This assay is completed in 60 min.

DNA from 115 German Caucasians was extracted from whole blood according to standard procedures. Our study population consisted of 40 healthy blood donors and 75 patients from the intensive care unit of our hospital. The reliability of the proposed assay was confirmed by restriction enzyme digestion with SfaNI. PCR was performed in disposable capillaries (Roche Diagnostics) with a reaction volume of 20 [micro]L containing 2 [micro]L of DNA (20-80 ng), 0.5 [micro]M each of the primers (sense, 5'-TTA CTC TTT GTC AAG ACA TGC CA-3'; anti-sense, 5'-ATG AGC CTC AGA CAT CTC CAG-3'), 2 [micro]L of reaction buffer [LightCycler fast start DNA master hybridization probes 10 X buffer (1X = 1.75 mM); Roche Diagnostics], 1 [micro]L of Mg[Cl.sub.2] (final concentration, 2.25 mM), and 0.2 [micro]M each of the labeled probes. The anchor probe (5'-CTA AGC TGC ACT TTT CCC CCT AGT-3') was labeled at the 3' end with fluorescein. The sensor probe, which is specific for the G allele (5'-GTG TCT TGC GAT GCT AAA GGA-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[degrees]C for 10 min, followed by 45 cycles of denaturation (95[degrees]C for 10 s, 20[degrees]C/ s), annealing (53[degrees]C for 10 s), and extension (72[degrees]C for 10 s). The melting curve consisted of one cycle at 95[degrees]C for 0 s and 45[degrees]C for 90 s, and then increasing the temperature to 85[degrees]C at a rate of 0.1[degrees]C/s. The fluorescence signal (F2) was monitored continuously during the temperature ramp and then plotted against the temperature (T). These curves were transformed to derivative melting curves [-d(F2)/dT vs T].

Representative results for the three different genotypes (GG, GC, and CC) are shown in Fig. 1. Of the 115 samples tested, 33% were GG, 46% were GC, and 21% were CC. In a larger study (383 Caucasians), the most frequent genotype was GC, followed by GG and CC (3). In a direct method comparison, our proposed new technique and the restriction enzyme technique with SfaNI gave identical genotyping results (data not shown). We conclude that the assay is rapid and accurate and seems especially suited for laboratories that process large numbers of samples.

We thank Andreas Nitsche and Olfert Landt (TIB MOLBIOL, Berlin, Germany) for designing the hybridization probes and reading the manuscript.

References

(1.) Whicher JT, Evans SW. Cytokines in disease. Clin Chem 1990;36:1269-81.

(2.) Ferrari SL, Garnero P, Emond S, Montgomery H, Humphries SE, Greenspan SL. A functional polymorphic variant in the interleukin-6 gene promoter associated with low bone resorption in postmenopausal women. Arthritis Rheum 2001; 44:196-201.

(3.) Fishman D, Faulds G, Jeffery R, Mohamed-Ali V, Yudkin JS, Humphries S, Woo P. The effect of novel polymorphisms in the interleukin-6 (IL-6) gene on IL-6 transcription and plasma IL-6 levels, and an association with systemic onset juvenile chronic arthritis. J Clin Invest 1998;102: 1369-76.

(4.) Rauramaa R, Vaisanen SB, Luong LA, Schmidt-Trucksass A, Penttila IM, Bouchard C, et al. Stromelysin-1 and interleukin-6 gene promoter polymorphisms are determinants of asymptomatic carotid artery atherosclerosis. Arterioscler Thromb Vasc Biol 2000;20:2657-62.

Thomas Bertsch [1] *

Wilma Zimmer [1]

Wendy Casarin [1]

Christof Denz [2]

Michael Quintel [2]

Klaus Fassbender [3]

Departments of [1] Clinical Chemistry,

[2] Anesthesiology, and [3] Neurology

University Hospital Mannheim

of the University of Heidelberg

Theodor-Kutzer-Ufer 1-3

D-68167 Mannheim, Germany

* Author for correspondence. Fax 49621-383-3819; e-mail thomas.bertsch@ ikc.ma.uni-heidelberg.de.
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Title Annotation:Letters
Author:Bertsch, Thomas; Zimmer, Wilma; Casarin, Wendy; Denz, Christof; Quintel, Michael; Fassbender, Klaus
Publication:Clinical Chemistry
Article Type:Letter to the editor
Date:Oct 1, 2001
Words:714
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