1. Two RANTES gene polymorphisms and their haplotypes in patients with myocardial infarction from two Slavonic populations Two RANTES gene polymorphisms and their haplotypes in patients with myocardial infarction from two Slavonic populations Petrek M 1,Tereshchenko I 1,3, Petrkova J 2, Lukl J 2, Mrazek F 1, Voevoda MI 3 1 Laboratory of Immunogenomics, Palacky University; 2 Dept. of Internal Medicine I, Faculty Hospital Olomouc, Czech republic 3 Inst. of Internal Medicine, RAMS Novosibirsk, Russian Federation This work was supported by Czech government (ME-856). I.T. is recipient of Visegrad fund fellowship)

2. Atherosclerosis, Inflammation & RANTES Inflammation of coronary artery wall is a critical process in the pathogenesis of myocardial infarction (MI). The chemokine RANTES/CCL5 activates and attracts T-lymphocytes to inflammatory sites. RANTES detected in T-cells from atherosclerotic lesions RANTES gene contains functional polymorphisms thus representing candidate for MI susceptibility.

3. RANTES variants & Myocardial infarction A single nucleotide polymorphism (SNP) locates at the position -403 of the RANTES gene promoter (Guanin to Adenin -403 G→A) RANTES-403*A allele tended to associate with a higher risk of coronary artery disease (CAD) in Hungarian patients (Szalai et al. Atherosclerosis 2001;158:233-39). In German population RANTES 403*A allele was significantly associated with CAD (Simeoni et al. European Heart Journal 2004; 25,1438-46) Another SNP is located in intron of the RANTES gene (In1.1 T/C). This SNP was not studied in coronary atherosclerosis.

4. AIMS To investigate whether RANTES promoter -403 G/A and intronic In1.1 T/C polymorphisms are associated with myocardial infarction in two Caucasian populations (Czech, West-Slavonic; Russian, East-Slavonic). In this way, to replicate previous reports on -403 SNP association with coronary atherosclerosis from other populations and to extend the study to another SNP in the RANTES gene.

5. Study population Diagnosis of myocardial infarction was made according to standard international criteria. (Eur Heart J. 2000;21:1502) Slavonic population West-Slavonic Czech 224 patients with MI 140 unrelated healthy controls East-Slavonic Russian 244 patients with MI 197 unrelated healthy controls

6. RANTES polymorphism genotyping and statistical analysis RANTES -403 G/A (rs2107538) and In1.1 T/C (rs2280789) polymorphisms were genotyped by polymerase chain reaction with sequence specific primers (PCR-SSP) Statistics: Statistical calculations were performed using the SPSS v.13.0 (SPSS Inc, Chicago, IL). The distribution of the RANTES genotypes and alleles were analyzed using the Pearson’s 2x2 contingency table  2 - test and odds ratios and 95% confidence interval (CI) were estimated. The control populations was tested for conformity to the Hardy-Weinberg equilibrium. Multiple variable logistic regression model with adjustment for conventional risk factors was used. Haplotypes were estimated by maximum likehood (software ARLEQUIN, v.3.000).

7. RESULTS Both SNPs from all groups in Hardy-Weinberg equilibrium There were no significant differences in RANTES -403 G/A genotype, allelic and phenotype (carriage) frequencies between MI patients and controls for both populations ( Tab.1) Also, no differences were observed for RANTES In1.1 T/C SNP( Tab.2) Multivariate analysis with combined data from both popula- tions showed no evidence of association, also after stratifica- tion by gender and age at 1 st infarction episode (data not shown) There were no differences between RANTES haplotypes in patients and control subjects (data not shown) The proportion of RANTES-403*A allele was similar in control groups across several Caucasian populations (Figure)

8. Table 1: Genotype, allele and phenotype (carriage rates) frequencies of the RANTES -403 G/A polymorphism in the groups of patients with myocardial infarction and control subjects (in two investigated populations) RANTES -403 G/A Czech population (N=364)Russian population(N=441) MI (N=224) Control (N=140) MI (N=244) Control (N=197) Genotype % GG 69.565.763.567.0 GA 27.831.429.928.9 AA 2.72.9 a 6.64.1 a Alleles % G 83.081.478.581.5 A 16.518.5 b 21.518.5 b A allele carriage % A 30.534.3 c 36.533.0 c a p value for genotype: Czech – 0.75, Russian – 0.48; b p value for alleles: Czech – 0.49, Russian – 0.27; c p value for A allele carriage: Czech – 0.45, Russian – 0.46.

9. Table 2: Genotype, allele and phenotype (carriage rates) frequencies of the RANTES In1.1 T/C polymorphism in the groups of patients with myocardial infarction and control subjects (in two investigated populations) RANTES Intron1.1 T/C Czech population (N=364)Russian population(N=441) MI (N=224) Control (N=140) MI (N=244) Control (N=197) Genotype % TT 82.078.670.872.6 TC 16.221.425.525.8 CC 1.803.71.6 Alleles % T 90.289.384.685.5 C 9.810.715.414.5 A allele carriage % C 17.921.427.027.4 p values for all comparisons > 0.05

10. Figure: Proportions of RANTES -403*A allele in patient with coronary artery disease and control groups across selected populations Czech republic, Olomouc; Russian Federation (RF), Novosibirsk: myocardial infarction, data of present study. Germany:coronary atherosclerosis, Simeoni et al.,Eur Heart J.,2004;25:1438. Hungary:coronary atherosclerosis, Szalai et al., Atherosclerosis.2001;158:233.

11. CONCLUSIONS RANTES promoter and intronic polymorphisms, and their haplotypes, are not associated with susceptibility to myocardial infarction and do not act as disease modifiers. Further, our data do not support association between RANTES -403*A allele and MI as previously reported from LURIC (LUdwigshafen Risk and Cardiovascular health) and Hungarian cohorts. At least in Slavonic population, RANTES promoter - 403 G/A and intronic In1.1 T/C polymorphism do not contribute to genetic determination of myocardial infarction.