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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.

Background— Cholesteryl ester transfer protein (CETP) inhibitors raise high-density lipoprotein (HDL) cholesterol, but torcetrapib, the first-in-class inhibitor tested in a large outcome trial, caused an unexpected blood pressure elevation and increased cardiovascular events. Whether the hypertensive effect resulted from CETP inhibition or an off-target action of torcetrapib has been debated. We hypothesized that common single-nucleotide polymorphisms in the CETP gene could help distinguish mechanism-based from off-target actions of CETP inhibitors to inform on the validity of CETP as a therapeutic target. Methods and Results— We compared the effect of CETP single-nucleotide polymorphisms and torcetrapib treatment on lipid fractions, blood pressure, and electrolytes in up to 67 687 individuals from genetic studies and 17 911 from randomized trials. CETP single-nucleotide polymorphisms and torcetrapib treatment reduced CETP activity and had a directionally concordant effect on 8 lipid and lipoprotein traits (total, low-density lipoprotein, and HDL cholesterol; HDL2; HDL3; apolipoproteins A-I and B; and triglycerides), with the genetic effect on HDL cholesterol (0.13 mmol/L, 95% confidence interval [CI] 0.11 to 0.14 mmol/L) being consistent with that expected of a 10-mg dose of torcetrapib (0.13 mmol/L, 95% CI 0.10 to 0.15). In trials, 60 mg of torcetrapib elevated systolic and diastolic blood pressure by 4.47 mm Hg (95% CI 4.10 to 4.84 mm Hg) and 2.08 mm Hg (95% CI 1.84 to 2.31 mm Hg), respectively. However, the effect of CETP single-nucleotide polymorphisms on systolic blood pressure (0.16 mm Hg, 95% CI −0.28 to 0.60 mm Hg) and diastolic blood pressure (−0.04 mm Hg, 95% CI −0.36 to 0.28 mm Hg) was null and significantly different from that expected of 10 mg of torcetrapib. Conclusions— Discordance in the effects of CETP single-nucleotide polymorphisms and torcetrapib treatment on blood pressure despite the concordant effects on lipids indicates the hypertensive action of torcetrapib is unlikely to be due to CETP inhibition or shared by chemically dissimilar CETP inhibitors. Genetic studies could find a place in drug-development programs as a new source of randomized evidence for drug-target validation in humans.

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.

BACKGROUND Cardiac troponin testing is central to the diagnosis of acute myocardial infarction. We evaluated a sensitive troponin I assay for the early diagnosis and risk stratification of myocardial infarction. METHODS In a multicenter study, we determined levels of troponin I as assessed by a sensitive assay, troponin T, and traditional myocardial necrosis markers in 1818 consecutive patients with suspected acute myocardial infarction, on admission and 3 hours and 6 hours after admission. RESULTS For samples obtained on admission, the diagnostic accuracy was highest with the sensitive troponin I assay (area under the receiver-operating-characteristic curve [AUC], 0.96), as compared with the troponin T assay (AUC, 0.85) and traditional myocardial necrosis markers. With the use of the sensitive troponin I assay (cutoff value, 0.04 ng per milliliter) on admission, the clinical sensitivity was 90.7%, and the specificity was 90.2%. The diagnostic accuracy was virtually identical in baseline and serial samples, regardless of the time of chest-pain onset. In patients presenting within 3 hours after chest-pain onset, a single sensitive troponin I assay had a negative predictive value of 84.1% and a positive predictive value of 86.7%; these findings predicted a 30% rise in the troponin I level within 6 hours. A troponin I level of more than 0.04 ng per milliliter was independently associated with an increased risk of an adverse outcome at 30 days (hazard ratio, 1.96; 95% confidence interval, 1.27 to 3.05; P=0.003). CONCLUSIONS The use of a sensitive assay for troponin I improves early diagnosis of acute myocardial infarction and risk stratification, regardless of the time of chest-pain onset.

Background— Environmental and genetic correlates of inflammatory marker variability are incompletely understood. In the family-based Framingham Heart Study, we investigated heritability and candidate gene associations of systemic inflammatory biomarkers. Methods and Results— In offspring participants (n=3710), we examined 11 inflammatory biomarkers (CD40 ligand, C-reactive protein, intercellular adhesion molecule-1, interleukin-6, urinary isoprostanes, monocyte chemoattractant protein-1, myeloperoxidase, P-selectin, tumor necrosis factor-α, tumor necrosis factor receptor II, fibrinogen). Heritability and bivariate genetic and environmental correlations were assessed by Sequential Oligogenic Linkage Analysis routines in 1012 family members. We examined 1943 tagging single-nucleotide polymorphisms in 233 inflammatory pathway genes with ≥5 minor allele carriers using a general genetic linear model. Clinical correlates explained 2.4% (CD40 ligand) to 28.5% (C-reactive protein) of the variability in inflammatory biomarkers. Estimated heritability ranged from 10.9% (isoprostanes) to 44.8% (P-selectin). Most correlations between biomarkers were weak although statistically significant. A total of 45 single-nucleotide polymorphism-biomarker associations met the q -value threshold of 0.25. Novel top single-nucleotide polymorphisms were observed in ICAM1 gene in relation to intercellular adhesion molecule-1 concentrations (rs1799969, P =1.32�10 −8 ) and MPO in relation to myeloperoxidase (rs28730837, P =1.9�10 −5 ). Lowest P values for trans -acting single-nucleotide polymorphisms were observed for APCS with monocyte chemoattractant protein-1 concentrations (rs1374486, P =1.01�10 −7 ) and confirmed for IL6R with interleukin-6 concentrations (rs8192284, P =3.36�10 −5 ). Novel potential candidates ( APCS, MPO ) need to be replicated. Conclusions— Our community-based data support the relevance of clinical and genetic factors for explaining variation in inflammatory biomarker traits.

Aims Chronic kidney disease is associated with increased risk of cardiovascular disease. Cystatin C is a promising marker to reliably mirror renal function. The role of cystatin C in patients with coronary artery disease (CAD) and normal or mildly reduced kidney function is the subject of current investigation. Methods and results In 2162 patients, over the whole spectrum of CAD, baseline cystatin C concentrations were measured. Patients with an estimated glomerular filtration rate of ≤60 mL/min per 1.73 m2 ( n = 295) were excluded. In patients with complete follow-up information ( n = 1827), 66 cardiovascular deaths were registered during a median follow-up of 3.65 years. Logarithmically transformed, standardized cystatin C was associated with cardiovascular death [hazard ratio: 1.94, 95% confidence interval (CI): 1.59–2.37, P < 0.001]. A potential threshold effect was observed; patients in the upper quartile had a 3.87-fold (95% CI: 2.33–6.42; P < 0.001) risk of mortality compared with the pooled lower quartiles. This risk association remained robust after adjustment for potential confounders including classical risk factors and N-terminal pro B-type natriuretic peptide. Serum creatinine was not associated with the outcome in this group of patients with normal renal function. Conclusion Results of this prospective study show that cystatin C is a potent predictor of cardiovascular mortality beyond classical risk factors in patients with CAD and normal or mildly reduced kidney function.

Interleukin-18 (IL-18) is a key inflammatory molecule suspected of being involved in the etiology of cardiovascular diseases (CVD). Five single nucleotide polymorphisms (SNPs) capturing the common genetic variation of the IL-18 gene (tag SNPs) were genotyped in five European prospective CVD cohorts including 1933 cases and 1938 non-cases as part of the MORGAM Project. Not a single SNP was found associated with CVD. However, a significant (P=0.002) gene-smoking interaction was observed. In smokers, the -105T allele was more frequent in cases than in non-cases (0.29 vs 0.25) and associated with an increased risk of disease (odds ratio (OR)=1.25 (1.07-1.45), P=0.005), whereas the inverse relationship tended to be observed in non-smokers (OR=0.90 (0.78-1.02), P=0.131). The gene-smoking interaction was broadly homogenous across the cohorts and was also observed through haplotype analyses. In conclusion, using the concerted effort of several European prospective CVD cohorts, we are able to show that one IL-18 tag SNP interacts with smoking to modulate the risk of developing CVD.

Aims Identification and functional characterization of variants in the neutrophil elastase (ELA2) gene in cardiovascular disease. Methods From participants of the ECTIM (Etude Cas-Temoins sur l'infarctus du Myocarde) Study with myocardial infarction (Ml) 2082 chromosomes were genetically scanned; 990 patients with Ml and 904 controls were genotyped for the common polymorphisms G-761A and S173S (C4890A). Expression vectors for Ela2 variants were transiently transfected, followed by Northern and Western blot analyses. Promoter variants were analyzed by transfection/reporter gene assays. Results We identified 11 genetic variants, two in the 5'-flanking (G-761A, -852/del53bp), six in exons (R49H, N81N, G93V, S173S, D222Y, P228L) and three in introns (C+29/in3T, C+149/in3T, C+137/in4T). In Belfast, 4890A allele carriers had a risk for Ml with an odds ratio (OR) of 1.44 (95% Cl 1.12-1.86; P= 0.005), the OR for Ml associated with the - 761 G/-4890A haplotype with reference to -761G/-4890C amounting to 2.38 (95% Cl 1.23-4.57; P=0.01). Transcript or protein expression of both allelic constructs (4890A and 4890C) did not, however, differ. Conversely, transcriptional activity was significantly elevated (

In our preceding studies, we have identified microsatellite polymorphisms inside the PSMA6 gene and in its 5′ upstream region. Following the observed associations of microsatellite polymorphisms with non-insulin dependent diabetes mellitus and Graves’ disease, we extended the evaluation of PSMA6 genetic variations to cardiovascular disorders and non-insulin dependent diabetes mellitus. New polymorphisms in the promoter region and exon 6 of the gene were identified by direct sequencing of the promoter region and all seven exons of the gene in 30 individuals of the European descent. Two SNPs at positions −110 and −8 from the translation start, in the promoter region and 5′ UTR, respectively, were analyzed. Neither polymorphism was associated with the risk of myocardial infarction. No significant association of the polymorphisms with plasma lipid levels or BMI was observed. A borderline association of both polymorphisms with diastolic blood pressure was observed in the control group. Genotype −8CG was significantly more frequent in type 2 diabetes patients, and haplotype C−110/G−8, compared to C−110/G−8 was associated with a higher risk of NIDDM.

Background: Concentrations of cholesterol, triglycerides and glucose are higher in young men with a paternal history of premature myocardial infarction than in age- and sex-matched controls. Aim: To test the hypothesis that insulin resistance constitutes the biological expression of increased coronary risk in these subjects. Design: A total of 407 male university students with a paternal history of premature myocardial infarction (cases) and 415 age- and sex-matched controls were investigated for differences in insulin sensitivity. Methods: Four methods of assessing insulin sensitivity were used: (i) insulin and glucose responses to an oral glucose tolerance test (OGTT); (ii) insulin and glucose responses to an oral fat tolerance test (OFTT); (iii) minimal modelling of insulin and glucose data from a frequent sample intravenous glucose tolerance test performed on a subset of 55 cases and 50 controls and (iv) homeostasis model assessment (HOMA) of insulin resistance. Results: The OFTT glucose response discriminated between cases and controls, with a smaller fall in glucose in cases compared with controls. The negative area under the glucose curve (AUC) (mean [standard error of the mean (SEM)]) was -1.42 (0.09) mmol min/L in cases and -1.76 (0.09) in controls ( P = 0.004). Peak height (mean [SEM]) was -0.65 (0.02) mmol/L in cases and -0.73 (0.02) in controls ( P = 0.007). The insulin responses were similar in cases and controls. Insulin AUC (mean [SEM]) was 161 (10) mU min/L in cases and 148 (10) in controls ( P = 0.34). This combination of findings suggests that insulin-stimulated glucose uptake was reduced in the cases. These findings were consistent across European regions. None of the other methods revealed any differences between cases and controls. Conclusion: In young men with a paternal history of myocardial infarction, an OFTT detects altered insulin sensitivity that is not identified by an OGTT, minimal modelling or HOMA.

The QT interval (QT) reflects cardiac ventricular repolarization and varies according to various known factors such as heart rate, gender and age. Nevertheless, a high intrasubject stability of the QT-RR pattern also suggests that a genetic component contributes to individual QT length. To determine whether single nucleotide polymorphisms (SNPs) in genes encoding cardiac ion channels were associated with the heart-rate corrected QT (QTc) length, we analyzed two groups of 200 subjects presenting the shortest and the longest QTc from a cohort of 2,008 healthy subjects. A total of 17 polymorphisms were genotyped; they were all in the Hardy-Weinberg equilibrium in both groups. Neither allele nor haplotype frequencies of the 10 KCNQ1 SNPs showed a significant difference between the two groups. In contrast, KCNH2 2690 C (K897T) and SCN5A 5457 T (D1819D) minor alleles were significantly more frequent in the group with the shortest QTc interval, whereas KCNE1 253 A (D85N), SCN5A 1673 G (H558R) and 1141-3 A minor alleles were significantly more frequent in the group with the longest QTc interval. Interestingly, an interaction was also found between the KCNH2 2690 A>C SNP and the KCNQ1 2031+ 932 A>G SNP suggesting that the effect of the KCNH2 2690 C allele on QTc length may occur within a particular genetic background. This suggests that genetic determinants located in KCNQ1, KCNE1, KCNH2 and SCN5A influence QTc length in healthy individuals and may represent risk factors for arrhythmias or cardiac sudden death in patients with cardiovascular diseases.

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.

Summary A detailed exploration of all the polymorphisms in candidate genes is required to better characterize the relationship between gene variability and complex traits. We propose a novel strategy for investigating the association between a highly polymorphic gene and a phenotype, by combining a multilocus genotype analysis and an haplotype analysis. For the multilocus genotype analysis, a data mining tool - termed DICE (Detection of Informative Combined Effects) ‐ was developed to identify the best subset of polymorphisms that are associated - individually or in combination ‐ with the phenotype. For the haplotype analysis, we used our recently developed method of haplotype-phenotype association to determine the most informative and parsimonious haplotype model fitting the data. We illustrate this strategy by investigating the association between twelve polymorphisms of the APOB gene and plasma apoB levels in 1442 European subjects. After exploring all main effects and interactions between polymorphisms, DICE identified the N4311S polymorphism as the most informative polymorphism in relation to apoB levels. Haplotype analysis led to the same conclusion. Additionally, DICE identified the E4154K (EcoRI) and the T2488T (XbaI) polymorphisms as potentially interesting. This selection was not modified by inclusion of the common APOE polymorphism in the analysis.

There is a growing body of evidence suggesting that the relationships between gene variability and common disease are more complex than initially thought and require the exploration of the whole polymorphism of candidate genes as well as several genes belonging to biological pathways. When the number of polymorphisms is relatively large and the structure of the relationships among them complex, the use of data mining tools to extract the relevant information is a necessity. Here, we propose an automated method for the detection of informative combined effects (DICE) among several polymorphisms (and nongenetic covariates) within the framework of association studies. The algorithm combines the advantages of the regressive approaches with those of data exploration tools. Importantly, DICE considers the problem of interaction between polymorphisms as an effect of interest and not as a nuisance effect. We illustrate the method with three applications on the relationship between (1) the P-selectin gene and myocardial infarction, (2) the cholesteryl ester transfer protein gene and plasma high-density-lipoprotein cholesterol concentration, and (3) genes of the renin-angiotensin-aldosterone system and myocardial infarction. The applications demonstrated that the method was able to recover results already found using other approaches, but in addition detected biologically sensible effects not previously described.

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.

Background Haemochromatosis is a common genetic disorder, inherited as an autosomal recessive trait that results in a progressive accumulation of iron in most tissues of the body. Positive association studies have been recently published between cardiovascular diseases and heterozygosity for the major mutation C282Y in the haemochromatosis gene HFE. Methods In the present work, we have determined the HFE genotypes for C282Y and H63D in subjects from two case-control studies: the ECTIM and GENIC studies, designed to identify genetic variants associated with myocardial and brain infarction, respectively. In addition, we tested whether HFE mutations were associated with the degree of arteriosclerosis assessed non-invasively by Doppler ultrasonography on the carotid and femoral arteries, in a group of apparently healthy individuals (the AXA Study). Results The prevalence of 282Y, and 63D allele carriers, did not differ between cases and controls in the ECTIM and in the GENIC studies, while 63D but not 282Y carriers were more numerous among subjects with atherosclerotic plaques in the AXA Study. Conclusions These three studies do not provide consistent evidence supporting the hypothesis that HFE mutations are associated with an increased risk of cardiovascular disease and with the development of arteriosclerosis.

Background Interleukin-10 (IL-10) is a cytokine with anti-inflammatory and B-cell-stimulating activity. IL-10 is expressed in human atherosclerotic plaques and recent studies have shown the involvement of IL-10 in the atherosclerotic process. Therefore, we hypothesized that polymorphisms in the IL-10 gene might be associated with a predisposition to coronary heart disease. Materials and methods To identify new polymorphisms in the human IL-10 gene, the entire coding sequence and the 3′ flanking sequence of the gene were screened by polymerase chain reaction–single strand conformation polymorphism (PCR-SSCR ) followed by sequencing. The polymorphisms identified, and three others which have been previously described in the promoter region of the IL-10 gene (G-1082A, C-819T, C-592A), were then investigated in the ECTIM Study, a large population-based case–control study of myocardial infarction. Results Four new polymorphisms were identified: one in exon 1 (G+78/ex1A), which predicts a Glycine to Arginine change at position 15 in the putative signal peptide of the protein, two in the intron 3 (C+19/in3T, T+953/in3C) and one in the 3′ flanking region (C+117T). All the IL-10 polymorphisms were in complete or nearly complete pairwise linkage disequilibrium. No case–control difference was found in genotype or allele frequencies for any of the polymorphisms. Conclusions Our results suggest that IL-10 polymorphisms are not associated with an increased risk of myocardial infarction.

Background Findings relating an association between an insertion/deletion polymorphism of the angiotensin converting enzyme (ACE) gene and myocardial infarction (MI) have been mixed. While other loci, such as the angiotensin II type-I receptor (AT 1 R), may modulate risk, few studies have adequately documented the risk in women. We aimed to study whether the findings in respect of ACE and AT 1 R in UK men were borne out in women. Methods Cases of MI (305 women, 391 men) in Belfast and Glasgow have been compared to controls (291 women, 356 men). These new samples augment the original men (200 cases, 181 controls) included from Belfast in the ECTIM study. Results Among men, the odds ratio for MI for ACE (DD vs. ID + II) was 1.03 (0.79, 1.34) and among women, 0.69 (0.47, 1.01). This heterogeneity between the risks in men and women was significant in Glasgow (P = 0.02). Among men and women the odds ratio for MI for AT 1 R (CC vs. AC + AA) was 1.02 (0.71, 1.47). There was a small gradient in risk, such that the odds ratio for DD genotype was 0.86 (0.63, 1.17) among subjects homozygous for the common AT 1 R alelle (AA): 0.94 (0.67, 1.30) among heterozygotes and 1.21 (0.53, 2.77) among CC subjects; but this interaction was not statistically significant. Conclusions Some of the contradictory findings concerning the ACE polymorphism and the risk of MI may be due to heterogeneity in the risk between men and women. The AT 1 R 1196 polymorphism is not an independent risk factor for MI in either sex.

Early growth response factor (EGR)-1 may play an important role in the development of atherosclerosis by inducing the expression of several relevant genes which contribute to the complex modulation of vascular structure and function, leading to vascular occlusive lesions. To investigate the possible role of molecular variants in the human EGR-1 gene for the predisposition to atherosclerosis or coronary heart disease we screened the 5'- and 3'- flanking regions and the entire coding sequence for polymorphisms by polymerase chain reaction/single-strand conformation polymorphism analysis and sequencing. Male patients (n=615) with myocardial infarction and 720 age-matched, male control subjects of the Etude Cas-Témoin de l'Infarctus du Myocarde were genotyped for two newly identified polymorphisms in the 5'- (C-151T) and 3'- (T+861C) flanking region of the EGR-1 gene using hybridization with allele-specific oligonucleotides. Allele and genotype frequencies did not significantly differ between patients with myocardial infarction and control subjects without coronary heart disease. In controls not taking hypolipidemic drugs there was a significant association of the -151T allele with lower plasma levels of total cholesterol (P=0.029), low-density lipoprotein cholesterol (P=0.025) and apolipoprotein B (P=0.038) and a higher ratio of high-density to low-density lipoprotein (P=0.049) than with the C-151 allele. We conclude that the C-151T polymorphism of the EGR-1 gene may contribute to modifications of the lipid metabolism. Our findings need to be replicated in independent studies, and in vitro promoter studies should evaluate the functional consequence of the -151T allele, which disrupts a consensus core sequence for the ubiquitous transcription factor activator protein 4.

Background Idiopathic dilated cardiomyopathy is a frequent cause of heart failure, a major concern of public health. Although idiopathic dilated cardiomyopathy may be familial, most cases are sporadic and the disease is considered to be multifactorial, for which genetic factors may account for a significant part. Methods and Results We hypothesized that genetic abnormalities of the endothelin pathway may be involved in idiopathic dilated cardiomyopathy pathophysiology and therefore examined the possible association between idiopathic dilated cardiomyopathy and polymorphisms in genes encoding endothelin 1, endothelin type A and type B receptors, in a case-control study (433 patients and 400 age- and sex-matched control subjects). Analysis of the Exon 8 C/T polymorphism in the endothelin receptor type A gene indicated that individuals who are homozygote for the T allele were at significantly increased risk for the disease (odds ratio: 1·9; 95% confidence interval: 1·2 to 3·01; P

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.

BACKGROUND: Tumour necrosis factor-alpha (TNF-alpha) is a cytokine that has multiple functions. Through its effects on lipid metabolism, coagulation, insulin resistance and endothelial function, TNF-alpha could be involved in cardiovascular pathophysiology. Given this possibility, we hypothesized that polymorphisms of the TNF-alpha gene might be associated with a predisposition to coronary heart disease (CHD). METHODS: The entire coding region and 1053 bp upstream of the transcription start site of the TNF-alpha gene were screened for polymorphisms using polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) and sequencing. Five polymorphisms were identified: four were located in the upstream region at positions -857, -851, -308, -238 from the first transcribed nucleotide and one was found in a non-translated region at position +691. Six-hundred and forty-one patients with myocardial infarction (MI) and 710 control subjects from the ECTIM Study were genotyped. RESULTS: The genotype frequencies were similar in cases and control subjects in the high-risk population of Belfast and in France; however, the TNF-alpha/-308A allele was more frequent in Belfast than in France (0.242 vs. 0.157; P < 0.0001), and carriers of this allele were more frequently obese than non-carriers [1.52 (1.15-1.99), P < 0.004]. No associations were found for the other polymorphisms. CONCLUSIONS: These results suggest that polymorphisms of the TNF-alpha gene are unlikely to contribute to CHD risk in an important way, but the TNF-alpha/-308 polymorphism should be investigated further in relation to obesity.

Segregation analysis using a regressive model with age- and sex-dependent effects was applied to family data of weight, fat mass (FM) and fat-free mass (FFM) to investigate the major gene hypothesis. The sample consisted of 220 nuclear families from the 'Stanislas Cohort' who volunteered for a free health examination (n = 913). FM and FFM were assessed by bioelectrical impedance. The data were adjusted for height2 and height prior to analysis. The spouse, father-offspring, mother-offspring and sib-sib correlations were: 0.16, 0.18, 0.25 and 0.32 for weight; 0.13, 0.20, 0.23 and 0.28 for FM; 0.18, 0.16, 0.29 and 0.41 for FFM. For the three phenotypes, models specifying a major gene with age- and sex-dependent effects and residual family correlations was better supported than models including only family correlations. For weight, the most parsimonious genetic model was a codominant model with a sex-dependent effect in parents and an age-increasing effect in offspring. For FM, the most parsimonious model was also a codominant model with sex-dependent effects in parents indicating higher effects in women than in men. For FFM, the most parsimonious model was a recessive model with no significant age or sex interaction, although the age interactions paralleled those observed on weight in offspring. For weight and FM, mendelian transmission was rejected. For FFM, the Mendelian and the environmental hypotheses were nearly equally supported and none was rejected when compared to general transmission. Then, evidence for a single major gene could not be inferred for any of the traits. This does not preclude the existence of several genes acting in a more complex way. However, our findings emphasize that weight is a composite phenotype reflecting different components which evolve in distinct ways during life span. For this reason, FM should be highly preferred to weight or BMI for the research of susceptibility genes to obesity.

Complement factor H (CFH) plays a critical role in the protection of host cells and tissues from damage by complement activation and has been suggested to protect against the progression of atherosclerosis. A polymorphism in the CFH gene, Y402H, known to be strongly associated with age-related macular degeneration, has been analyzed in relation to coronary artery disease (CAD) in several studies with conflicting results. We investigated the association of polymorphisms of the CFH gene in two large-scale studies on CAD and myocardial infarction (MI). The AtheroGene Study included a cohort of cases with CAD (n = 1,303) prospectively followed for a median period of 6.2 years, among whom198 experienced a cardiovascular event, and a group of 483 control subjects. The AtheroGene Study population was genotyped for the Y402H, I62V, and E936D polymorphisms. There was no significant difference in genotypic or allelic frequencies between CAD cases and controls. Among cases, no significant association was found with prospective cardiovascular outcome. Many inflammatory proteins, including the C-reactive protein, were measured, and none of the polymorphisms showed an association with these markers. The Etude Cas-Temoin de l’Infarctus du Myocarde (ECTIM) Study compared 1,034 patients with MI and 1,039 controls from France and United Kingdom. The ECTIM Study population was genotyped for the Y402H polymorphism. Genotype and allele frequencies were similar in cases and controls. These results do not support an involvement of common nonsynonymous polymorphisms of the CFH gene in predisposition to CAD and its complications.

The impact of common variants in the apolipoprotein gene cluster ( APOC3-A4-A5 ) on prospective coronary heart disease (CHD) risk was examined in healthy UK men. Of the 2808 men followed over 9 years, 187 had a clinically defined CHD event. Examination of 9 single nucleotide polymorphisms (SNPs) in this group revealed that homozygotes for APOA4 S347 had significantly increased risk of CHD [hazard ratio (HR) of 2.07 (95%CI 1.04 to 4.12)], whereas men homozygous for APOC3 1100T were protected [HR 0.28 (95%CI 0.09 to 0.87)]. In stepwise multiple regression analysis, after entering all the variants and adjusting for established risk factors APOA4 T347S alone remained in the model. Using all nine SNPs, the highest risk-estimate haplotypes carried APOA4 S347 and rare alleles of the two flanking intergenic markers. The protective effect of APOC3 1100T could be explained by negative linkage disequilibrium with these alleles. To determine the association of APOA4 T347S with apoAIV levels, the relationship was examined in 1600 healthy young European men and women. S347 homozygotes had significantly lower apoAIV plasma levels (13.64±0.59 mg/dL) compared with carriers of the T347 allele (14.90±0.12 mg/dL) ( P =0.035). These results demonstrate that genetic variation in and around APOA4 , independent of the effects of triglyceride, is associated with risk of CHD and apoAIV levels, supporting an antiatherogenic role for apoAIV.

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 AvaI restriction site. The relationship between the -971G/A polymorphism, plasma lipid parameters and plasma CETP concentration was evaluated in the Etude Cas-Témoins 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.

OBJECTIVE--To assess the reliability of reported family histories of myocardial infarction. DESIGN--A case-control study in which reported histories of first degree relatives were validated from death certificates, general practitioners' records, and hospital notes. SETTING--Participants enrolled in the Belfast centre of the World Health Organisation's study monitoring trends and determinants in cardiovascular disease (MONICA). SUBJECTS--200 men who survived myocardial infarction and 200 age matched controls drawn randomly from the population. MAIN OUTCOME MEASURES--The sensitivity, specificity, positive predictive value, and proportion of overall agreement with validated records of reported family histories of myocardial infarction in first degree relatives; odds ratios for myocardial infarction, given at least one reported relative or at least one verified relative being affected. RESULTS--349 of the 400 probands provided detailed family histories, reporting on 2812 first degree relatives. The overall sensitivity, specificity, and positive predictive value of reported histories were 67.3%, 96.5%, and 70.5% for cases and 68.5%, 97.7%, and 73.8% for controls. The kappa coefficients were modest: 0.65 for cases and 0.68 for controls. The odds ratios for myocardial infarction, given at least one affected relative, were not substantially inflated by recall bias. Some recall bias was evident for the probands' reports of their siblings' histories of myocardial infarction, the odds ratio for a reported history being 1.67 (95% confidence interval 1.09 to 2.57) and for the validated history 1.54 (1.01 to 2.37). CONCLUSIONS--Although the relative risk of disease is correctly estimated, the predictive accuracy of a casual family history of myocardial infarction may limit the effectiveness of targeted screening programmes. They may, however, complement other strategies based on genetic testing.

OBJECTIVES--To improve blood pressure control among hypertensive ( > 140/90 mmHg) excessive alcohol drinkers. DESIGN--Fourteen worksite physicians were randomised onto an intervention group and a control group. The intervention was based on training the worksite physicians and follow up of those hypertensive subjects defined as excessive drinkers. Follow up was based on self monitoring of alcohol consumption by the subject, in view of the results of their gamma glutamyl transferase (GGT) activity determination. SETTING--Fourteen workplaces in France - mainly in the industrial sector. SUBJECTS--Altogether 15 301 subjects were screened by the 14 physicians: 129 of these were included in the study. MAIN OUTCOME MEASURES--This was the difference between the initial systolic blood pressure (SBP) and the SBP one year later (delta BP). Secondary criteria were the difference between the initial and final diastolic blood pressure (delta DBP) and delta BP at two years; antihypertensive treatment; state alcohol consumption (delta AC); delta GGT; and body mass index (delta BMI). RESULTS--The decrease in SBP levels was significantly larger in the intervention group than in the control group: at one year, delta SBP values were -11.9 (15.6) mmHg and -4.6 (13.8) respectively (p < 0.05). This benefit was still observed after two years of follow up (-13.8 (17.4) mmHg v -7.5 (14.2) mmHg (p < 0.05)). No difference was observed in DBP. The percentage of treated subjects did not differ between groups. At one year, delta AC was larger in the intervention group (-2.8 (5.2) U/d) than in the control group (-1.6 (3.4) (p < 0.1)). delta GGT and delta BMI did not differ between the two groups. A weak positive correlation was observed between delta AC and delta SBP (r = 0.16). CONCLUSION--An intervention aimed at the hypertensive excessive drinkers in a working population was found to be effective in reducing SBP on a long term basis (two years). The mechanisms of reduction in alcohol consumption and improved drug compliance cannot be ascertained in this pragmatic study. From a public health point of view, reducing the excess cardiovascular risk among a "hard to reach" population seems feasible with a strategy specifically designed for this high risk group.

The hereditary haemochromatosis gene (HFE) has recently been proposed as a disease modifying gene.1 The rationale is that two common mutations of the HFE gene (C282Y and H63D) are found in a majority of patients with genetic haemochromatosis who are either homozygotes (C282Y/C282Y) or compound heterozygotes (C282Y/H63D). These mutations have been shown to contribute to more subtle modifications of iron homeostasis at the heterozygous state.2 In turn, iron may predispose to myocardial damage through the production of activated oxygen species. Recently, Mahon and colleagues have reported an association between the H63D mutation and idiopathic dilated cardiomyopathy (IDCM).3 In this study, 207 unrelated white patients with dilated cardiomyopathy and 200 controls were tested for HFE C282Y and H63D mutations. An increased proportion of H63D heterozygotes was found among patients (36%) as compared to the control group (27%). No association was found with C282Y mutation and as the H63D mutation had a relatively minor effect on iron status, these authors proposed that this association may be …

Inter-individual variability in telomere length is highly heritable and has been correlated with risk of coronary heart disease (CHD). Our aim was to determine the association of mean leukocyte telomere length with paternal history of premature myocardial infarction (MI). Mean leukocyte telomere length was measured with real-time polymerase chain reactions in 369 male students (18–28 years) with a paternal history of MI before the age of 55, recruited from 14 European universities, serving as cases and 396 age-matched controls with no paternal history of CHD. Overall, cases had borderline significantly shorter mean length (~550 bp), adjusted for age and geographical region, than controls (p = 0.05). A significant difference in telomere length across the geographical regions of Europe was observed (p

The aim of this study was to investigate the influence of APOA5 variants on fasting lipids and to the response to both an oral fat tolerance test (OFTT) and an oral glucose tolerance test (OGTT). The association of two APOA5 SNPs [S19W (SNP5), −1131T>C (SNP3)] and an APOA4/A5 intergenic SNP [−12238T>C (SNP4)] were examined in healthy young men (n=774) who had undergone both an OFTT and an OGTT. Both −1131T>C and S19W rare alleles were associated with triglyceride (TG)-raising effects (11%, P=0.008; 21% (in cases), PC rare allele was associated with higher waist to hip ratio (P

Background Interleukin-18 is a pro-inflammatory cytokine suspected to be associated with atherosclerosis and its complications. We had previously shown that one single nucleotide polymorphism (SNP) of the IL18 gene was associated with cardiovascular disease (CVD) through an interaction with smoking. As a further step for elucidating the contribution of the IL-18 pathway to the etiology of CVD, we here investigated the association between the genetic variability of two IL-18 receptor genes, IL18R1 and IL18RAP, with the risk of developing CVD. Methods Eleven tagging SNPs, 5 in IL18R1 and 6 in IL18RAP, characterizing the haplotypic variability of the corresponding genes; were genotyped in 5 European prospective CVD cohorts including 1416 cases and 1772 non-cases, as part of the MORGAM project. Both single-locus and haplotypes analyses were carried out to investigate the association of these SNPs with CVD. Results We did not find any significant differences in allele, genotype and haplotype frequencies between cases and non-cases for either of the two genes. Moreover, the search for interactions between SNPs located in different genes, including 5 IL18 SNPs previously studied in the MORGAM project, and between SNPs and environmental factors remained unfruitful. Conclusion Our analysis suggests that the variability of IL18R1 and IL18RAP genes are unlikely to contribute to modulate the risk of CVD.