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ObjectivesEarly identification of myocardial infarction in chest pain patients is crucial to identify patients at risk and to maintain a fast treatment initiation.BackgroundThe aim of the current investigation is to test whether determination of copeptin, an indirect marker for arginin-vasopressin, adds diagnostic information to cardiac troponin in early evaluation of patients with suspected myocardial infarction.MethodsBetween January 2007 and July 2008, patients with suspected acute coronary syndrome were consecutively enrolled in this multicenter study. Copeptin, troponin T (TnT), myoglobin, and creatine kinase-myocardial band were determined at admission and after 3 and 6 h.ResultsOf 1,386 (66.4% male) enrolled patients, 299 (21.6%) had the discharge diagnosis of acute myocardial infarction, 184 (13.3%) presented with unstable angina, and in 903 (65.2%) an acute coronary syndrome could be excluded. Combined measurement of copeptin and TnT on admission improved the c-statistic from 0.84 for TnT alone to 0.93 in the overall population and from 0.77 to 0.9 in patients presenting within 3 h after chest pain onset (CPO) (p < 0.001). In this group the combination of copeptin with a conventional TnT provided a negative predictive value of 92.4%.ConclusionsIn triage of chest pain patients, determination of copeptin in addition to troponin improves diagnostic performance, especially early after CPO. Combined determination of troponin and copeptin provides a remarkable negative predictive value virtually independent of CPO time and therefore aids in early and safe rule-out of myocardial infarction.

We aimed at associating common osteopontin (OPN) gene variants with cardiovascular disease phenotypes.We scanned the OPN gene in 190 chromosomes from myocardial infarction (MI) patients and identified five variants in the promoter, three synonymous and one non-synonymous variant. All variants were investigated in case–control studies for MI (ECTIM: 990 cases, 900 controls) and brain infarction (BI) (GENIC: 466 cases, 444 controls). Promoter variants were functionally analyzed by bandshift assays, the coding D147D [T/C] by Western blot. Allele D147D C was independently and significantly associated with lower apoB levels ( P =0.044 [ECTIM] P =0.03 [GENIC]), its allele frequency was significantly lower in patients with BI compared to controls (OR [95% CI] 0.39 [0.20–0.74], P =0.004), and C allele carriers had a significantly lower frequency of presence of carotid plaques ( P =0.02). Bandshifts with HepG2 and Ea.hy926 nuclear proteins did not reveal any functionality of promoter variants, whereas the OPN-441C-containing construct resulted in reduced OPN protein expression in Western blots, complying with its potential protective effect on the phenotypes studied.We here provide evidence that a portion of the OPN locus is likely to associate with cardiovascular disease-related phenotypes. However, further experiments are warranted to clarify the functional role of OPN variants.

Summary. Background: Thrombin activatable fibrinolysis inhibitor (TAFI) attenuates fibrinolysis. Results on the association between TAFI levels and the risk of coronary artery disease (CAD) are inconsistent. Objectives: We investigated the association between TAFI levels and the risk of cardiovascular events in CAD. Patients/Methods: 1668 individuals with angiographically proven CAD at baseline were followed for a median of 2.3 years, as part of the prospective AtheroGene cohort. Fifty-six deaths from cardiovascular (CV) causes and 35 non-fatal CV events were observed. Results: At baseline, three TAFI measurements were available: one evaluating the total amount of TAFI (t-TAFI), one measuring the TAFIa/TAFIai amount, and the last the released activated peptide (TAFI-AP). TAFIa/TAFIai levels were associated with increased risk of CV death [hazard ratio (HR) for one tertile increase, 2.38 (1.56–3.63); P

Insulin resistance is polygenic in origin, and can be observed at an early age. We have shown that variations in APOC3−482T>C and hepatic lipase (LIPC)-514C>T), individually (APOC3 alone) and interactively, modulate insulin and glucose levels after an OGTT in young healthy men participating in the European Atherosclerosis Research Study II (EARSII). Variation in the insulin gene (INS) variable number tandem repeat (VNTR) has been found to predispose to type 1 and type 2 diabetes. We have evaluated the HphI site 23 bp upstream of the INS gene (a surrogate marker for the VNTR) in EARSII (n=822), to determine if variation in INS contributes to insulin resistance. Carriers of the INS VNTR class III (HphI−) allele (frequency=0.29 (95%CI 0.27, 0.31)) had significantly higher 60-min insulin concentrations after the OGTT (P=0.014) and a marginally higher AUC insulin (P=0.07), compared to class I (HphI+) homozygotes. However, this effect on AUC insulin was modified by the level of physical activity, displaying significant gene:environment interaction (P=0.03). We tested for gene:gene interaction between the INS VNTR and both the LIPC−514C>T and APOC3−482T>C. While there was a significant interaction between INS VNTR and LIPC−514C>T on AUC glucose (P=0.013) and on AUC insulin (P=0.015), there was no interaction with APOC3−482T>C. Thus, despite a modest effect of the INS VNTR alone, the influence of this variant on insulin sensitivity was modified by gene:environment and gene:gene interactions, illustrating the biological complexity of insulin resistance.

There is accumulating evidence for a role of tumor necrosis factor-α (TNF-α) in insulin resistance induced by obesity. The purpose of this study was to investigate whether the TNFα/G-308A polymorphism was associated with responses to oral glucose and fat tolerance tests in a case–control study comparing male offspring with a paternal history of premature myocardial infarction (cases, n =335) to age-matched controls ( n =340) recruited from 14 European university populations. Genotype frequencies did not significantly differ between cases and controls. Among cases, those carrying the A allele exhibited a higher area under the curve for insulin (64.5 vs 55.9 mU h/l, P =0.009), a higher increment between baseline concentration and peak of insulin (63.1 vs 52.8 mU/l, P =0.005) and a greater decrease between peak and insulin at 120 min (49.1 vs 36.8 mU/l, P =0.003) than those with the GG genotype. No such effect was observed in control subjects. No association was observed with response to a fat tolerance test either in cases or in controls. The present results suggest that the TNFα/G-308A polymorphism might interact with other susceptibility factors to coronary heart disease to predispose to insulin resistance, and that the ability of TNF-α to induce insulin resistance may extend beyond obesity.

Human paraoxonase-1 is hypothesised to protect serum lipoproteins from oxidative stress. Decreased serum activity of paraoxonase-1 in animal models is associated with an increased risk of vascular disease and has been linked to the anti-oxidant capacity of the enzyme. Promoter polymorphisms of the human paraoxonase-1 gene strongly influence serum concentrations of the enzyme. The present study examined the hypothesis that promoter polymorphisms may be genetic risk factors for vascular disease in man. Genotypes arising from the promoter C(-907)G polymorphism were analysed in the ECTIM2 population. The global odds ratio for myocardial infarction, comparing the high expressor GG genotype to other genotypes, was 0.77 (0.61–0.97) ( P =0.024). The association with the promoter genotype was more pronounced in the youngest age group (odds ratio 0.52 (0.31–0.87), P =0.012) and was progressively lost with age (respectively 50 years to P =0.26; >60 years, P =0.45). There was no association between the promoter genotypes and serum lipids. The data are consistent with the high expressor promoter genotype being linked to reduced risk of myocardial infarction. The influence of the genotype may be compromised in older patients.

The plasma cholesteryl ester transfer protein (CETP) plays a key role in reverse cholesterol transport (RCT) by mediating the transfer of cholesteryl ester (CE) from high-density lipoprotein (HDL) to atherogenic ApoB-containing lipoproteins, including VLDL, IDL and LDL. We describe a new polymorphism located at position −971 in the human CETP gene promoter, which corresponds to a G/A substitution at a potential Ava I restriction site. The relationship between the −971G/A polymorphism, plasma lipid parameters and plasma CETP concentration was evaluated in the Etude Cas-Temoins de l'Infarctus du Myocarde (control–myocardial infarction cases) cohort, and revealed that the −971G/A polymorphism (A allele frequency: 0.491) was significantly associated with both plasma high-density lipoprotein cholesterol (HDL-C) levels and CETP concentration ( P =0.006 and 0.009, respectively). Subjects with genotype −971GG displayed both low HDL-C levels and high plasma CETP concentration, while genotype −971AA subjects displayed the inverse relationship. Evaluation of potential interactions between the −971G/A and the −629C/A or TaqIB polymorphisms demonstrated that the −971G/A polymorphism interacts significantly with the functional −629C/A site and the TaqIB polymorphism with respect to plasma HDL-C levels ( P =0.0014 and 0.012, respectively), but does not affect plasma CETP concentration. These results clearly suggest that the interaction between the 971G/A polymorphism and either the −629C/A or the TaqIB polymorphism on plasma CETP concentration is different than that implicated in HDL-C levels. Transient transfection of HepG2 cells revealed that the −971G/A polymorphism did not modulate transcriptional activity of the human CETP gene promoter. The −971G/A promoter polymorphism therefore constitutes a non-functional marker. Furthermore, the observed effects of the −971G/A polymorphism on both plasma CETP concentration and HDL-C levels are due to functional variants in linkage disequilibrium with it. Our findings strongly suggest the existence of as yet unidentified functional polymorphisms in the CETP gene promoter that could explain the association between specific polymorphisms of the CETP gene and both plasma HDL-C and CETP concentrations.

OBJECTIVES The study investigated the potential role of eight candidate genes in the susceptibility to idiopathic dilated cardiomyopathy (IDC). BACKGROUND Idiopathic dilated cardiomyopathy has a familial origin in 20% to 25% of cases, and several genetic loci have been identified in rare monogenic forms of the disease. These findings led to the hypothesis that genetic factors might also be involved in sporadic forms of the disease. In complex diseases that do not exhibit a clear pattern of familial aggregation, the candidate gene approach is a strategy widely used to identify susceptibility genes. All genes coding for proteins involved in biochemical or physiological abnormalities of cardiac function are potential candidates for IDC. METHODS We studied 433 patients with IDC and 401 gender- and age-matched controls. Polymorphisms investigated were the I/D polymorphism of the angiotensin I-converting enzyme (ACE) gene, the T174M and M235T polymorphisms of the angiotensinogen (AGT) gene, the A-153G and A+39C polymorphisms of the angiotensin-II type 1 receptor (AGTR1) gene, the T-344C polymorphism of the aldosterone synthase (CYP11B2) gene, the G-308A polymorphism of the tumor necrosis factor-alpha (TNF) gene, the R25P polymorphism of the transforming growth factor beta1(TGFB1) gene, the G+11/in23T polymorphism of the endothelial nitric oxide synthase (NOS3) gene and the C-1563T polymorphism of the brain natriuretic peptide (BNP) gene. RESULTS None of the polymorphisms were significantly associated with the risk or the severity of the disease. CONCLUSIONS We did not find evidence for an involvement of any of the 10 investigated polymorphisms in the susceptibility to IDC.

Summary Two strategies involving whole-genome association studies have been proposed for the identification of genes involved in complex diseases. The first one seeks to characterize all common variants of human genes and to test their association with disease. The second one seeks to develop dense maps of single-nucleotide polymorphisms (SNPs) and to detect susceptibility genes through linkage disequilibrium. We performed a molecular screening of the coding and/or flanking regions of 36 candidate genes for cardiovascular diseases. All polymorphisms identified by this screening were further genotyped in 750 subjects of European descent. In the whole set of genes, the lengths explored spanned 53.8 kb in the 5′ regions, 68.4 kb in exonic regions, and 13 kb in the 3′ regions. The strength of linkage disequilibrium within candidate regions suggests that genomewide maps of SNPs might be efficient ways to identify new disease-susceptibility genes, provided that the maps are sufficiently dense. However, the relatively large number of polymorphisms within coding and regulatory regions of candidate genes raises the possibility that several of them might be functional and that the pattern of genotype-phenotype association might be more complex than initially envisaged, as actually has been observed in some well-characterized genes. These results argue in favor of both genomewide association studies and detailed studies of the overall sequence variation of candidate genes, as complementary approaches.

The genetic factors that underlie idiopathic dilated cardiomyopathy (IDCM) have not yet been elucidated. Since beta1-adrenoceptors are downregulated in patients with IDCM, and since beta-blocker therapy is consistently beneficial in this setting, we hypothesized that genetic variation in the beta1-adrenoceptor might affect susceptibility to and/or severity of IDCM. As no intragenic polymorphism was available, a systematic screening of the gene was first performed. The organization and sequence of the human beta1-adrenoceptor gene were established using polymerase chain reaction, single-strand conformation polymorphism analysis and sequencing. The gene comprises 1434 bp and no intron was observed. We found a unique and frequent polymorphism (C1165G) which predicts an Arg389Gly substitution. The association of this polymorphism with IDCM was then analysed using the PCR-restriction fragment length polymorphism method in the CARDIGENE population, a clinically well-characterized population of IDCM. Genotypic distribution was in agreement with Hardy-Weinberg equilibrium. There were no differences in the beta1-adrenoceptor allele frequencies between IDCM (n=426; C/G=0.76/0.24) and age- and sex-matched control subjects (n=395; C/G=0.78/0.22). Within the patient group, no association was observed with the severity of the disease. In conclusion, the genomic organization of beta1-adrenoceptor is described here for the first time. We found a unique and frequent polymorphism in the coding sequence of the gene. No association was observed between IDCM and the genetic variant. Its possible involvement in other cardiac diseases related to the beta1-adrenoceptor remains to be analysed.

A common polymorphism has been described in the methylenetetrahydrofolate reductase (MTHFR) gene, substituting an alanine (A) for a valine (V), where the V allele results in a thermolabile enzyme with reduced activity. This polymorphism is easily detectable by PCR amplification and digestion with HinfI restriction enzyme. We describe the use of the MADGE high throughput genotyping system for rapid typing of this polymorphism. Seven hundred and eighty five individuals participating in the European Atherosclerosis Research Study II (EARS II), aged 22-25 from 14 universities in 12 countries across Europe were genotyped for this polymorphism. The frequency of the V allele was 0.32 overall (95% CI; 0.30-0.35), but was significantly lower in the Baltic countries (0.23; 95% CI; 0.19-0.28) compared with the other regions of Europe (0.37; 95% CI; 0.32-0.38) (P < 0.001). Individuals homozygous for the V allele had statistically significant (P < 0.001) higher plasma homocysteine (16.5 micromol/l) compared with those heterozygous for an A allele (10.4 micromol/l) or homozygous for an A allele (10.0 micromol/l). This effect was seen in all countries and regions of Europe. Mean plasma homocysteine levels were significantly higher in the South compared to the Baltic, UK and Middle regions (P = 0.001), but this difference was not explained by the difference in the frequency of the V allele in the samples. This polymorphism explained 12.3% of the total sample variance in plasma homocysteine, other measured factors (smoking, alcohol consumption, systolic blood pressure, physical activity) explained 0.7%. This study demonstrates the large and consistent impact of the thermolabile MTHFR variant on plasma homocysteine levels in different European populations, and shows a regional difference in the levels of homocysteine that must be explained by other genetic or environmental factors.

Using polymerase chain reaction (PCR) based techniques, we have identified individuals in the ECTIM study of myocardial infarction survivors (cases) and healthy matched controls who are carriers for a mutation of the gene for lipoprotein lipase (LPL) which alters amino acid 9 from aspartic acid to asparagine (LPL-D9N). The frequency of carriers in the cases from Belfast and France (3 separate centres) was 2.5 and 3.7%, respectively (mean 3.3%, 95% CI 1.9-4.7) and in the controls 2.0 and 2.9%, respectively (mean 2.7%, 95% CI 1.6-3.8%), but this difference was not statistically significant. In the cases, carriers of the allele for LPL-N9 had higher levels of several plasma lipid traits including total triglycerides (TG) (30%), very low density lipoprotein (VLDL) cholesterol (19%), apo E (24%), apo C-III (17%), lipoprotein particles (Lp) containing both apo E and apo B (LpE:B) (32%), and particles containing both apo C-III and apo B (LpCIII:B) (39%), and this effect was consistent in cases both from Belfast and from the French centres combined. By contrast, in the controls there were no differences in any lipid trait between carriers and non-carriers of the mutation that was consistent between the French centres and Belfast. There were no significant differences in the levels of any measured factor between cases and controls that could explain the different effect on plasma lipid traits associated with the mutation. However, compared to the non-carriers, in both cases and controls who carried the mutation, plasma TG concentrations were higher in those whose body mass index (BMI) was above the mean of the sample (26.0 kg/m2), with statistically significant interaction seen between BMI and genotype and levels of apo C-III, and lipoprotein particles containing both apo C-III and apo B (P < 0.02). The data suggest that carriers for the LPL-N9 mutation have a mild genetic predisposition to developing hyperlipidaemia and an atherogenic lipid profile, but that this requires the presence of other genetic or environmental factors for full expression, one of which appears to be increasing obesity.

The levels of different lipid, lipoprotein and haemostatic variables were assessed in 615 control subjects of the ECTIM Study, defined by five groups of alcohol consumption: non-drinkers, 0or = 15 g/day, 15.or = 36 g/day, 36.or = 66 g/day and66 g/day. After adjustment for age, body mass index, cigarette consumption and country, alcohol consumption was associated with an increase in HDL-cholesterol (0.47 +/- 0.02 to 0.59 +/- 0.01 g/l in non-drinkers and66 g/day consumers, mean +/- S.E.M., P0.0001), apolipoproteins A-I and A-II (1.37 +/- 0.03 to 1.60 +/- 0.03 g/l and 0.32 +/- 0.01 to 0.41 +/- 0.01 g/l, respectively, P0.0001), LpA-I, LpA-I:A-II (0.46 +/- 0.01 to 0.50 +/- 0.01 g/l and 0.75 +/- 0.02 to 0.91 +/- 0.02 g/l, respectively, P0.001) and PAi-1 activity (134 +/- 11 to 177 +/- 11 U/ml, P0.001). Conversely, no increases were found for total and LDL-cholesterol, triglycerides, apolipoproteins B and C-III, LpE:B, LpC-III:B, fibrinogen and factor VII. Hence, among the lipid and haemostatic variables studied, only HDL parameters and PAi-1 activity were increased by alcohol consumption.

Human secreted phospholipases A2 (sPLA2s) represent novel attractive therapeutic targets and biomarkers in coronary artery diseases (CAD). We have shown that human Group X sPLA2 (hGX sPLA2) is present in atherosclerotic lesions and that hGX sPLA2 modified LDL induces foam cell formation. To elucidate whether hGX sPLA2 has a causative role in CAD we have screened the human PLA2G10 gene to identify frequent polymorphisms, and we have examined their possible association with cardiovascular end-points and intermediate inflammatory phenotypes in a large prospective study of patients with CAD (the AtheroGene study). Although no significant association was found between the various polymorphisms identified and lipids or inflammatory markers, patients carriers of the C allele of the T-512C polymorphism located in the 5’ untranslated region showed a decreased risk of recurrent cardiovascular events. Molecular analysis of the only missense variant (R38C) showed its functional relevance as it leads to a profound change in expression and secretion of hGX sPLA2.

In a European study an insertion (/)/deletion (D) polymorphism in the angiotensin converting enzyme (ACE) gene has been shown to be associated with the risk of myocardial infarction (Ml). In the same study, we investigated the association of the polymorphism with a parental history of fatal MI. There was an excess of both DD (odds ratio 2·6, p=0·02) and ID (odds ratio=1·9, p=0·08) genotypes among those having a parental history of Ml, confirming that genetic variation in the ACE locus could be involved in the risk of MI.