Over a three year span (2004 to 2006), a total of 976 unrelated male patients younger than 65 years were enrolled into a study due to a symptomatic ACS with STEMI as well as NSTEMI. This prospective multi-centre registry involved four heart centres and six cardiologic departments in Germany. The study was approved by the ethical committee of the University Witten/Herdecke, Germany, and conformed to the declaration of Helsinki. All participants gave written informed consent to participation.
MI was defined as follows: ischemic type chest pain lasting for more than 20 minutes, at least 0.1 mV of ST-segment elevation in the limb leads and/or at least 0.2 mV elevation in the precordial leads and one of the following criteria: left bundle branch block, new Q wave (at least 0.03 s), elevated creatine kinase or positive troponin T or I. All cases underwent cardiac catheterization and interventional or surgical revascularization. Patients < 18 and > 65 years, with unstable angina or without central European origin were excluded from further studies.
Blood samples and DNA preparation
EDTA-blood samples were drawn from each participant at baseline ward round. Genomic DNA was extracted from 350 μl of these samples using the BioRobot EZ1 and the EZ1 blood extraction kit according to the manufacturer's instructions (QIAGEN; Hilden, Germany). DNA was quantified using the BioPhotometer (Eppendorf; Hamburg, Germany) and each sample was diluted to a final concentration of 25 ng/μl.
PCR amplification and genotyping
Genotyping of the investigated SNPs (rs1333049, rs1333040, rs10757274, rs2383206, rs10757276, and rs2383207) was carried out by real time PCR and subsequent melting curve analysis on a LightCycler 480 instrument (Roche Applied Science; Mannheim, Germany). The primers and hybridization probes were designed and synthesized by Tib MolBiol GmbH (Berlin, Germany). PCR was carried out in 96-well plates (Roche Applied Science; Mannheim, Germany) using 12.5 ng of genomic DNA as template in a final reaction volume of 5 μl. Reaction mixtures contained 0.5 μM of each primer, 0.15 μM of SNP-specific hybridization probes and 1 μl of LightCycler 480 Genotyping Master (Taq DNA polymerase, reaction buffer, 15 mM MgCl2, and a dNTP mixture with UTP instead of dTTP) (Roche Diagnostics GmbH, Mannheim, Germany).
The cycling program consisted of 10 minutes of initial denaturation at 95°C, followed by 35 cycles (rs1333049, rs2383206, rs2383207, rs10757274, rs10757278) or 40 cycles (rs1333040) of denaturation at 95°C for 5 seconds, annealing at 53°C (rs1333049, rs2383206, rs2383207), at 59°C (rs1333040) or at 62°C (rs10757274, rs10757278) for 10 seconds, and extension at 72°C for 10 seconds. After PCR melting curves were generated by holding the reaction mixture at 95°C for 1 minute, stepwise lowering of the temperature to 65°C, 55°C and 45°C, holding for 30 seconds at each temperature, lowering to 40°C for 2 minutes, followed by continuously heating to 75°C. Melting curve analyses were conducted using the LightCycler 480 software according to the manufacturer's instructions (Roche Diagnostics GmbH; Mannheim, Germany).
The distribution of genotypes and the allelic frequencies were investigated and analyzed by alignment to previously published data of applicable controls. Genotypes were tested for Hardy-Weinberg equilibrium among MI cases and controls using a chi-square test with one degree of freedom. Differences in the genotype distribution were tested for statistically significance and odds ratios (ORs) were determined using the 2-way contingency table chi-square test using a freely accessible program (http://statpages.org/ctab2x2.html
). For all data, the association was considered to be significant for p < 0.05.