Comparison of Methods for Determination of Interleukin 6 G(-174)C Promoter Polymorphism

Interleukin 6 (IL-6) is one of the key proinflammatory cytokines that is produced by many different cell types, including cells of the immune system, endothelial cells, fibroblasts, myocytes, and adipocytes. It can mediate inflammatory but also stress-induced responses (10,14–16). IL-6 induces the secretion of monocyte chemotactic protein, an important mediator of inflammatory events in atherosclerosis, and regulates the expression of adhesion molecules and the release of cytokines such as TNF-α and IL-1β (3,13). IL-6 basal and peak levels show inter-individual variability determined in part by genetic factors (4,12). Recently, a G-to-C polymorphism at nucleotide position (174) in the promoter region of the IL-6 gene has been identified by Fishman et al. (9) and has been associated with an altered transcriptional response to stimuli such as endotoxin or IL-1 (8). In some studies, individuals carrying the GG genotype have been reported to show higher plasma levels for IL-6 in comparison to carriers of the CC genotype (5,8). However, controversial data with higher IL-6 plasma concentrations in individuals homozygous for the CC genotype have also been described (1). This polymorphism has been associated with systemic-onset juvenile chronic arthritis (8), osteoporosis (8), atherosclerosis (17), the incidence and outcome of sepsis (19), susceptibility to type 1 diabetes mellitus (11), coronary heart disease (1), or IL-6 levels after coronary artery bypass surgery (4). To clarify, if and how the polymorphism in the promoter region of the IL6 gene influences plasma levels and the predisposition to various common diseases, studies on large cohorts of patients and control individuals are required. Thus, fast, reliable, and economical assays for determination of the IL-6 promoter polymorphism have been developed and described in the literature. However, up to now no direct comparison of the different assays in regard to reliability and economic aspects has yet been performed. We compared different methods, PCR followed by restriction enzyme digestion, a newly developed mutagenically separated PCR, and real-time PCR using fluorescence-labeled hybridization probes, for their reliability, the required working time, and reagent costs. For all tests, DNA from 50 individuals was extracted from venous blood anticoagulated with EDTA using the MagNA Pure LC isolation system (Roche Applied Science, Mannheim Germany). All individuals had given their informed consent to DNA analysis. PCR amplification for restriction enzyme digestion was carried out following a published protocol (7) with primers forward 5′-TGACTTCAGCTTTACTCTTTGT-3′ and reverse 5′-CTGATTGGAAACCTTATTAAG-3′ (Tib Molbiol, Berlin, Germany). PCR was performed in an Eppendorf® cycler in a final volume of 50 μL containing approximately 50 ng DNA, 1.5 mM MgCl2, 200 μM each dNTP (Amersham Bioscience, Piscataway, NJ, USA), 10 pmol each primer, and 1.25 U AmpliTaq Gold® (Applied Biosystems, Foster City, CA, USA). DNA was amplified for 35 cycles of denaturation for 40 s at 95°C, annealing for 40 s at 55°C, and elongation for 40 s at 72°C. A final extension for 5 min at 72°C completed the reaction. PCR amplification was followed by restriction digestion with the enzyme SfaNI (New England Biolabs, Hertfordshire, UK) at 37°C for 3 h. The digested products were electrophoretically separated on 6% precast polyacrylamide Novex® gels (Invitrogen, Carlsbad, CA, USA) for 50 min at 160 V. Gels were stained with SYBR Green® (1:10 000; Molecular Probes, Eugene, OR, USA). Individuals carrying the GG genotype showed bands at 140 and 58 bp, individuals with the heterozygous GC genotype had bands at 198, 140, and 58 bp, and individuals homozygous for the CC genotype had one band at 198 bp. The new allele-specific PCR method developed by us is based on the principle of mutagenically separated PCR (18), a single-tube PCR technique with allele-specific primers differing in length by 9 bp. Base mismatches in the allele-specific primers introduce differences into the PCR products that minimize cross-reactions of the products in DRUG DISCOVERY AND GENOMIC TECHNOLOGIES