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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.

Despite extensive exploration of many genes, strong evidence of a molecular genetic association with coronary heart disease (CHD) or myocardial infarction (MI) remains to be obtained. Recently, significant interest has emerged in mapping genetic susceptibility for complex traits through whole-genome studies association generating promoting data that will determine the genetic contribution to common human diseases such as coronary heart disease. The aim of the present case-control study including 324 healthy controls and 296 patients with coronary heart disease from Tunisia, was to assess relation between three polymorphisms previously reported to be strongly associated with coronary heart disease in the Welcome Trust Case Control Consortium (WTCCC) and the German myocardial infarction family studies: locus 9p21.3 (rs 1333049), locus 6q25.1 (rs6922269) and 2q36.3 (rs2943634). By single locus analysis, no differences in genotype distribution and allelic frequency were found between the two groups of study. The risk allele (C) for rs2943634 was less frequent among Tunisian population than in controls from the WTCCC and German studies (57% vs 65%). The three SNPs previously reported to be associated with CHD were not replicated in our small sample.

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.

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

P-selectin plays a key role in inflammation and atherosclerosis, and polymorphic variants of P-selectin were implicated in the pathogenesis of atherosclerotic and inflammatory changes, including coronary heart disease (CHD) in many ethnic groups. We investigated the contribution of P-selectin promoter (-2123C/G, -1969G/A) and exon (Ser290Asn, Asn562Asp, Thr715Pro) polymorphisms to CHD genetic susceptibility among 298 Tunisian CHD patients and 339 controls. Minor allele and genotype frequencies of the five P-selectin SNPs were comparable between patients and controls, except for -2123G/G genotype which was more frequent in cases. The 715Pro allele was present at lower frequency in Tunisians than in Europeans, and was not protective of CHD. Linkage disequilibrium was seen between -1969G/A, and both Ser290Asn and Asn562Asp. Five-loci haplotype analysis did not identify any CHD-protective or CHD-susceptible haplotypes. To our knowledge, this was the first case-control study to be performed on an Arab/North-African population, and demonstrates that none of the five P-selectin polymorphisms investigated influence CHD susceptibility in Tunisian Arabs.

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.

Reduced glomerular filtration rate defines chronic kidney disease and is associated with cardiovascular and all-cause mortality. We conducted a meta-analysis of genome-wide association studies for estimated glomerular filtration rate (eGFR), combining data across 133,413 individuals with replication in up to 42,166 individuals. We identify 24 new and confirm 29 previously identified loci. Of these 53 loci, 19 associate with eGFR among individuals with diabetes. Using bioinformatics, we show that identified genes at eGFR loci are enriched for expression in kidney tissues and in pathways relevant for kidney development and transmembrane transporter activity, kidney structure, and regulation of glucose metabolism. Chromatin state mapping and DNase I hypersensitivity analyses across adult tissues demonstrate preferential mapping of associated variants to regulatory regions in kidney but not extra-renal tissues. These findings suggest that genetic determinants of eGFR are mediated largely through direct effects within the kidney and highlight important cell types and biological pathways. J.T. and P.H. are consultants for Servier. J.C. received research grants and honoraria from Servier. K.S. obtained research support from Boehringer Ingelheim. The remaining authors declared no competing financial interests.

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.

Objective The SAH gene locus has recently been proposed to be involved in obesity-related hypertension in Japanese individuals. Methods To replicate independently the initial findings in another ethnic group, we scanned the entire SAH gene in 190 Caucasian chromosomes. A total of 651 patients with essential hypertension and 776 controls (PEGASE Study) were genotyped for all identified variants using allele-specific oligonucleotides, and single nucleotide polymorphism as well as haplotype analyses were carried out. We also performed transient transfection experiments, northern and western blots, immunoprecipitation, and acyl-coenzyme A synthetase activity assays. Results We identified five polymorphisms in the promoter region (C−1808T, G−1606A, −962ins/del, G−451A, T−67C), two in introns 5 and 7 (T+9/In5C, A+20/In7T), and one missense variant (K359N). Carriage of the −1606A allele was significantly associated with hypertension [odds ratio (OR) 1.28, P = 0.049] as was 359N (OR 1.35, P = 0.048) compared with non-carriers. Conversely, for −962del, the OR for hypertension was 0.80 (P = 0.042). The SAH alleles −1606A and 359N, but not −962ins/del, displayed a raising effect on body mass index (BMI; P = 0.004 and P = 0.030, respectively) in hypertensive as well as in control individuals. After adjustment for BMI in hypertensive individuals, only the OR associated with −962ins/del remained significant (OR 0.77, P = 0.028). Functional analyses in BHK did not reveal differences for SAH 359N or 359K-containing constructs, formally excluding K359N as the functional variant. Conclusion We confirm recent evidence that the SAH locus is associated with obesity-related hypertension, in which pathophysiological context SAH variants affecting blood pressure remain, however, to be shown.

Objective— To get a better insight into the role of hemostasis in coronary artery disease (CAD), we assessed the impact of von Willebrand factor (vWF), fibrinogen, thrombin-antithrombin (TAT) complexes, D-dimers, and plasmin-antiplasmin (PAP) complexes on the risk of cardiovascular event in a prospective cohort of CAD patients. Methods and Results— The prospective Athero gene cohort includes 1057 individuals with an angiographically proven coronary artery disease at baseline. After a median follow-up of 6.6 years, 135 individuals died from a cardiovascular cause and 97 had a nonfatal cardiovascular event. Higher levels of all 5 hemostatic markers at baseline were associated with an increased risk of cardiovascular death, but not of nonfatal event. Except for vWF, these associations remained significant after adjustment for conventional cardiovascular risk factors and C-reactive protein (CRP) levels ( P for trend according to increasing tertiles=0.20, 0.011, 0.026, 0.019, and 0.01 for vWF, fibrinogen, TAT, D-Dimer, and PAP, respectively). When including the 5 hemostatic markers in a stepwise Cox regression analysis where conventional risk factors and CRP were forced into the model, fibrinogen and D-dimers remained independently associated with the risk of cardiovascular death. Adjusted hazard ratios (95% CI) associated with one SD increase of fibrinogen and D-dimers were 1.27 (1.04 to 1.55) and 1.29 (1.09 to 1.53), respectively. Conclusions— In patients with coronary artery disease, fibrinogen and D-dimer levels are independent predictors of subsequent cardiovascular death. Our data support a role of impaired coagulation/fibrinolysis process in the complications of coronary artery disease.

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.

Gardner syndrome, a phenotypic variant of familial adenomatous polyposis, is characterized by the classical clinical triad of skin and soft tissue tumours, osteomas and intestinal polyposis, but disease patterns with pairs of these findings have also been reported. Different mutations in the adenomatous polyposis coli (APC) gene have been shown to be associated with Gardner syndrome disease phenotypes. A 36-year-old patient presented with multiple epidermal cysts on the face, left ear lobe and neck, and the possible diagnosis of Gardner syndrome was based on the additional findings of two classical osteomas in the left radius and ulna and a cold non-malignant nodule of the thyroid gland. Intestinal polyposis was lacking at the time of examination. Major deletions but not microdeletions were excluded by a cytogenetic analysis with 650 chromosomal bands per haploid set. Systematic sequencing of the entire coding region of the APC gene (> 8500 bp) of the patient and five healthy controls was also performed. As a results, new APC gene polymorphisms were identified in exons 13 [A545A (A/G)] and 15 [G1678G (A/G), S1756S (G/T), P1960P (A/G)]. We also detected D1822V (A/T) which has recently been reported to be potentially related to colorectal carcinoma, and genotyped 194 randomly chosen healthy individuals from the Glasgow area for this as well as for the above variants in exons 13 and 15. Interestingly, of the 194 controls, 112 carried the DD (57.7%), 71 the DV (36.6%), and the remaining 11 (5.7%), including our patient, the VV genotype. It is therefore unlikely that APC D1822V serves as an important marker for colorectal carcinoma. In conclusion, we failed to identify obvious germline candidate mutations in > 8500 bp of the coding region of the APC gene in a patient with multiple epidermal cysts, osteomas and a thyroid gland nodule; major chromosomal deletions were excluded. Therefore, we assume that only the presence of intestinal polyposis is a marker for Gardner syndrome.

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.

The angiotensin II type 2 (AT2) receptor is thought to play a role in cardiovascular disorders such as neointima formation after vascular injury, cardiac hypertrophy and myocardial infarction (MI). Recently, the biallelic polymorphism G + 1675A in intron 1 of the AT2 receptor gene has been associated with left ventricular posterior, septal and relative wall thickness, as well as left ventricular mass index in young hypertensive males.To investigate its potential role in left ventricular hypertrophy (LVH) and other cardiovascular traits, 1968 individuals from two population samples (the Glasgow Heart Scan, GLAECO and Glasgow Heart Scan Old, GLAOLD studies) with echocardiographically and electrocardiographically assessed phenotypes, were genotyped for G + 1675A using allele-specific oligonucleotide hybridization. Both studies had a similar design, only the age-ranges differed, being 25-74 years in the GLAECO study and 55-74 years in the GLAOLD study, so that internal consistency of results could also be assessed. Since the AT2 gene is located on the X chromosome, males and females were analysed separately.The + 1675A allele frequency was 0.49 and 0.51, in the GLAECO and GLAOLD studies, respectively. In both studies, the genotype frequencies were similar in hypertensive and non-hypertensive individuals. In the GLAOLD study, in females with episodes of coronary ischemia and MI, the AT2 + 1675A allele was more common than in females with no episode (86.5% vs. 73.5%, respectively; P0.007). This effect was not observed in males. In the same study, AT2 + 1675A allele carriers were more common in males with LVH, than in those without LVH (60.3% vs. 46.0%, respectively; P = 0.047). This result was unchanged after exclusion of subjects taking antihypertensive drugs (including ACE inhibitors) (64.4% vs. 47.4%, P = 0.038). However, in the GLAECO study, these results could not be replicated, even when subjects55 years of age were considered separately.Our study gives rise to a potential implication of the AT2 G + 1675A polymorphism in LVH and coronary ischemia subgroups. Since these results were not consistent in both studies, but are partially in agreement with two independent investigations, further efforts should be made to elucidate the specific nature of these genotype/phenotype interactions.

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.

Abstract— P-selectin is a cellular adhesion molecule that mediates the interaction of activated endothelial cells or platelets with leukocytes. Increased levels of soluble P-selectin have been reported in various cardiovascular disorders. We measured serum soluble P-selectin levels as well as 3 polymorphisms of the P-selectin gene (C-2123G, A-1969G, and Thr715Pro) in a large cohort of patients with documented coronary artery disease (n=869) and a healthy control group (n=334). The 3 P-selectin polymorphisms were strongly associated with P-selectin levels and altogether explained 7.3% and 18.6% of the P-selectin variability in patients and controls, respectively. Genotype distributions did not significantly differ between patients and controls. P-selectin levels were increased in patients younger than 55 years of age compared with controls (135.2 vs 114.3 ng/mL, P P

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.

Advanced glycation end-products (AGEs) may play an important role in the pathogenesis and progression of cardiovascular and renal complications of diabetes. Four putative AGE receptors (RAGEs), AGE-R1, AGE-R2, and AGE-R3 have been described. In this study, we scanned the sequence of the genes encoding these AGE receptors in 48 patients with type 1 diabetes and investigated the identified polymorphisms (n = 19) in 199 type 1 diabetic patients with nephropathy and 193 type 1 diabetic patients without nephropathy. Overall, none of the polymorphisms was strongly associated with nephropathy. The minor allele of a polymorphism located in the promoter region of the RAGE gene (C-1152A) conferred a weak protective effect (P < 0.05) and was associated with a longer duration of nephropathy-free diabetes (P = 0.08).

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.

Abstract —The matrix Gla protein (MGP) is an important inhibitor of vessel and cartilage calcification that is strongly expressed in human calcified, atherosclerotic plaques and could modulate plaque calcification and coronary heart disease risk. Using a genetic approach, we explored this possibility by identifying polymorphisms of the MGP gene and testing their possible association with myocardial infarction (MI) and plaque calcification. Eight polymorphisms were identified in the coding and 5′-flanking sequences of the MGP gene. All polymorphisms were investigated in 607 patients with MI and 667 control subjects recruited into the ECTIM Study (Etude Cas-Témoins de l’Infarctus du Myocarde) and in 717 healthy individuals with echographically assessed arterial calcification and atherosclerosis who were participating in the AXA Study. In the ECTIM Study, alleles and genotypes were distributed similarly in patients and controls in the whole study group; in only 1 subgroup of subjects defined as being at low risk for MI were the concordant A −7 and Ala 83 alleles more frequent in patients with MI than in controls ( P P MGP polymorphisms were not associated with any investigated clinical phenotype. Transient transfection experiments with allelic promoter-reporter gene constructs and DNA-protein interaction assays were carried out to assess possible in vitro functionality of the promoter variants detected at positions −814, −138, and −7 relative to the start of transcription. When compared with the −138 T allele, the minor −138 C allele consistently conferred a reduced promoter activity of −20% ( P P A −7 or Ala 83 alleles of the MGP gene may confer an increased risk of plaque calcification and MI; however, the observed relationships are weak or limited to subgroups of patients and therefore need confirmation.

Abstract —Tissue factor (TF) is a transmembrane protein considered to be responsible for the initiation of coagulation. TF gene expression may be induced in monocytes and endothelial cells and is present in atherosclerotic plaque to initiate thrombus formation. To investigate whether individual differences in TF gene expression could predispose subjects to thrombosis, we sequenced the 5′ domain of the gene up to nucleotide 2732 and found 6 different polymorphisms: 4 of them were completely concordant and defined 2 haplotypes with similar frequencies, designated as 1208 D and 1208 I. Genotyping of patients with myocardial infarction in a case-control study involving 2354 subjects showed no association between the polymorphisms and nonfatal coronary thrombosis. In another study involving 255 patients with venous thromboembolism and 1204 controls, allele D was less common in the cases ( P =0.022). The odds ratio associated with the presence of at least 1 D allele was 0.72 ( P =0.031). Comparison of subgroups of control subjects who were homozygous for the D or I allele demonstrated a lower plasma TF concentration in DD homozygotes. These results indicate that the TF gene promoter exists in 2 major forms differing at 4 sites. The 1208 D haplotype is not associated with coronary thrombosis but is associated with reduced plasma TF levels and a lower risk of venous thrombosis.

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.

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.

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.

Abstract —The allele and haplotype frequency of the –1654 C/T and –1641 A/G protein C (PC) gene promoter polymorphisms was determined and analyzed according to circulating PC concentrations in 394 healthy subjects aged 20 to 60 years. The CG haplotype was associated with a lower PC concentration in both homozygous and heterozygous subjects compared with noncarriers. The TA allele had the reverse effect, but only in homozygotes. The distribution of the CG and TA alleles was significantly different in 242 patients, aged 17 to 60 years, with venous thromboembolism. The CG allele increased the risk of thrombosis, with an OR of 1.39 (95% confidence interval (CI), 1.04 to 1.87). The TA allele was protective in subjects aged TA was also significantly associated with older age at the first thrombosis. This study confirms the link between the PC gene promoter and circulating PC levels, and suggests a complex effect on the risk of thrombosis.

Lifestyle, genetic, bioclinical and biochemical factors of European university students aged 18-26 years, with and without documented paternal histories of premature coronary heart disease, have been compared in the European Atherosclerosis Research Studies (EARS) I and II.To highlight consistencies and inconsistencies between findings in the two studies.All measurements were made according to strict protocols, by trained technicians using validated methods. The results for men in EARS I are compared with those from EARS II which was confined to men.In both studies we found no differences between cases and controls in lifestyle factors and bioclinical factors except that controls were taller. We found inconsistent differences between obesity indices and antecedents of arterial hypertension. In both studies we found consistent differences between cases and controls in levels of total cholesterol and apolipoprotein B, both levels being higher in cases. The lack of any difference between levels of high-density lipoprotein cholesterol and apolipoprotein A1 was also found consistently. Inconsistent differences were found for levels of triglycerides and apolipoprotein E. For most of the candidate genes that were studied, no differences between cases and controls were found, but different polymorphisms were associated with levels of lipids, apoproteins and fibrinogen independently of case-control status. Some of these associations were potentiated by lifestyle factors. The interaction between genetic and environmental factors is further illustrated with results from the association of apolipoprotein E polymorphism with the level of apolipoprotein B and a variety of other determinants of apolipoprotein B level.In the EARS studies a documented family history of premature coronary heart disease was mainly expressed in terms of biochemical factors that are determined both by nature and by nurture.

Abstract —A polymorphism at position 825 (C→T) of the cDNA that encodes the β3 subunit (GNB3) of the pertussis toxin–sensitive G protein was recently shown to be associated with human hypertension. To verify this finding and to investigate whether this polymorphism could also be associated with coronary heart disease, we analyzed the GNB3 variant in subjects from 2 previously described studies: Projet d’Etude des Gènes de l’hypertension Artérielle Sévère à modérée Essentielle (PEGASE), a case-control study of moderate to severe hypertension (681 cases and 308 controls), and Etude Cas-Témoins de l’Infarctus du Myocarde (ECTIM), a case-control study of myocardial infarction (MI) (564 cases and 633 controls). Genotyping was performed with allele-specific oligonucleotides. Genotype and allele frequencies were in Hardy-Weinberg equilibrium in all groups. Allele and genotype frequencies did not differ significantly between case patients with essential hypertension or MI and control subjects. In the ECTIM study, the 825T allele frequencies in cases and controls from Belfast, Northern Ireland, were 0.31 and 0.30 ( P =0.79), respectively; the corresponding frequencies in cases and controls from France were 0.33 and 0.31 ( P =0.30), respectively. In the PEGASE study, the 825T allele frequency was 0.35 in female and male cases and 0.31 in male normotensive controls ( P =0.12). The odds ratios for hypertension (PEGASE) and MI (ECTIM) associated with T-allele carrying were 1.23 (95% confidence interval, 0.94 to 1.62; P =0.13) and 1.11 (95% confidence interval, 0.88 to 1.39; P =0.37), respectively. There was no association of the GNB3 polymorphism with early onset of hypertension, familial history of hypertension, or blood pressure level. We conclude that the 825C/T polymorphism of the GNB3 gene did not contribute in any important way to the risk of essential hypertension or MI in these studies.

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.

Background —We investigated the relationship between variation in the apolipoprotein (apo) AI-CIII-AIV gene cluster and response to an oral glucose test (OGTT) and oral fat load test (OFTT) in the EARSII group of young, healthy male offspring whose fathers had had a myocardial infarction before the age of 55 years (cases, n=407) compared with age-matched controls (n=415). The apoCIII variations examined were C3238G ( Sst I) in the 3′-UTR, C1100T in exon 3, C-482T in the insulin response element (IRE), and T-2854G in the apoCIII-AIV intergenic region. Methods and Results —The postprandial response was regulated by variation at the T-2854G and C3238G sites. After the OFTT, carriers of the rare alleles had delayed clearance of triglyceride (Tg) levels; G-2854 carriers showed the largest effect on Tg (AUC, 24% greater, P P P P =0.03). However, after adjustment for fasting level of Tg, only the effect with the T-2854G remained significant. Variation at the C-482T (IRE) determined response to the OGTT, with carriers of the rare T-482 having significantly elevated glucose (28.7% AUC, P =0.013) and insulin (20.5% AUC, P Conclusions —These data suggest that specific genetic variants at the apoCIII gene locus differentially affect postprandial and response to OGTT and suggest a novel mechanism for the effects of variation at this locus on risk for atherosclerosis.

The aims of the study were to investigate associations of the apolipoprotein (apo) A-IV polymorphisms Thr347Ser and Gln360His with anthropomorphic measurements and fasting and postprandial lipids in subjects participating in the European Atherosclerosis Research Study II (EARS II). The allelic frequencies of Ser347 and His360 were 0.185 and 0.067, respectively, in the sample as a whole. There were no significant differences in rare allele frequency between cases (offspring of fathers who suffered a myocardial infarction before the age of 55 years) and controls. Control subjects who were carriers of Ser347 had significantly higher body mass indices (BMIs), waist:hip ratios, total and low density lipoprotein cholesterol and triacylglycerol (TG) concentrations (all P ≤ 0.02) than control subjects who were non-carriers, but these effects were not seen in the cases. Control subjects who were carriers of His360 had lower BMIs (P = 0.04), cholesterol and TG concentrations (both P ≤ 0.07) compared to non-carriers, but these effects were not seen in the cases. After consumption of an oral fat load, carriers of His360 who were most obese (subjects in the third tertile of BMI) had significantly reduced postprandial lipemia (P < 0.03, as assessed by area under the curve).—Fisher, R. M., H. Burke, V. Nicaud, C. Ehnholm, and S. E. Humphries. Effect of variation in the apoA-IV gene on body mass index and fasting and postprandial lipids in the European Atherosclerosis Research Study II. J. Lipid Res. 1999. 40: 287–294.

SummaryThe A4070G polymorphism in exon 13 of the factor V (FV) gene, which replaces His by Arg at position 1299 of the B domain, was recently shown to influence circulating FV levels and to contribute to the activated protein C (APC) resistance phenotype. We examined the impact of this polymorphism in a population of unselected patients with venous thromboembolic disease (VTE). The prevalence of the G4070 (R2) allele was determined in 205 patients and 394 healthy subjects of similar age and sex distribution. Thirty-seven patients (18%) were heterozygous for the R2 allele and 1 (0.5%) was homozygous. Forty-four controls (11.2%) were heterozygous for the R2 allele and 1 (0.2%) was homozygous. Thus, the allelic frequency was significantly higher in the patients with VTE than in the healthy controls, with respective values of 9.5% and 5.8%. The odds ratio was 1.8 (95% CI: 1.1-2.8, p = 0.02), pointing to an increased risk of VTE in carriers of the R2 allele. After excluding subjects with putative or confirmed gene defects (mainly the FV R506Q mutation), the R2 allele was still a risk factor for VTE in the remaining patients, with an odds ratio of 2.0 (95% CI: 1.2-3.5, p = 0.01), demonstrating that this polymorphism is itself a risk factor. This study also confirms that the R2 allele influences APC resistance (APCR) in the absence of the FV R506Q mutation.

The microsomal triglyceride transfer protein (MTP) catalyzes the transfer of triglyceride, cholesteryl ester, and phosphatidylcholine between phospholipid surfaces. The 97-kD subunit imparts lipid transfer activity and thus plays a role in the assembly of apolipoprotein B (apoB)-containing lipoproteins. We tested whether polymorphisms in the promoter region of the large subunit of the MTP gene might be related to different plasma lipid variables, atherosclerosis, and the risk of myocardial infarction (MI). We screened 838 bp in the promoter region of the MTP gene by PCR-SSCP and identified two polymorphisms at positions −400 (MTP/−400 (A→t)) and −164 (MTP/−164 (T→c)), the latter being situated on a putative sterol responsive element (SRE) consensus sequence. The two polymorphisms, investigated in 622 male patients with MI and in 728 age-matched controls participating in the ECTIM Study, were in nearly complete linkage disequilibrium (|D′| = ′0.98, less frequent alleles being preferentially associated, P < 0.001). There were no significant differences in genotype or allele frequencies between patients with MI and controls. Moreover, no significant associations between the two promoter polymorphisms and several lipid variables measured in the control groups of the ECTIM Study or coronary artery stenosis, angiographically assessed in patients with MI, were detected. We conclude that these MTP polymorphisms are unrelated to lipid variables or coronary heart disease in this study.—Herrmann, S-M., O. Poirier, V. Nicaud, A. Evans, J-B. Ruidavets, G. Luc, D. Arveiler, C. Bao-Sheng, and F. Cambien. Identification of two polymorphisms in the promoter of the microsomal triglyceride transfer protein (MTP) gene: lack of association with lipoprotein profiles. J. Lipid Res. 1998. 39: 2432–2435.

Cystathionine β synthase (CBS) is a key enzyme in homocysteine metabolism. We have examined four apparently non-functional polymorphisms in the CBS gene and have determined their frequency, degree of linkage disequilibrium and association with plasma homocysteine levels. The polymorphisms are a 68 bp insertion in exon 8, C699T in exon 8, C1080T in exon 11 and C1985T in the 3′ untranslated region. 785 individuals participating in the European Atherosclerosis Research Study II (EARSII), from 11 countries across Europe were genotyped for these polymorphisms. The 68bp insertion had the highest frequency in the UK and in the Middle region, with a lower frequency in the Baltic and the South (p=0.01), and the exon 11 polymorphism had the highest frequencies of the rare allele in the Baltic (p

Abstract —The H -allele of the intron 8 Hin dIII polymorphism in the lipoprotein lipase (LPL) gene has been associated with a lower risk of myocardial infarction (MI) and plasma levels of triglycerides (TG). To test whether the Hin dIII site was in linkage disequilibrium with the functional variant LPL Serine447Stop (S447X), subjects from the European Atherosclerosis Research Study (EARS I) were genotyped for both polymorphic sites. This study included 515 offspring of fathers with a premature (.99), with only three of the four possible haplotypes identified: H +S447, H− S447, and H− X447. The frequency of the H− X447 but not of the H− S447 haplotype was significantly lower in cases than in control subjects (.090 versus .117, P H +S447 haplotype, the odds ratio of having a paternal history of premature MI for H− X447 heterozygotes (≈20% of the population) was 0.71 (95% confidence interval, 0.55 to 0.92). In addition, there was an increase of the H− X447 haplotype frequency from north to south in control subjects (0.119 in Finland to 0.143 in the Mediterranean region, P H +S447 haplotype, the H− X447 haplotype was associated with significantly lower concentrations of plasma TGs (5.4% lower, P =.01), with this effect being consistent over the regions of Europe. There was no significant evidence for a heterogeneity of effect between males and females or between cases and control subjects, although the effect on TG levels appeared to be the greatest in male cases (11% lower, P =.05). In a second study (EARS II), of 332 cases and 342 control subjects, postprandial clearance of TGs after a standard fat meal was examined. The H− X447 haplotype was associated with significantly lower postprandial triglyceride levels than was the H +S447 haplotype (9.4% smaller area under the curve, P H allele appeared to be mainly mediated by the X447 mutation, and although the lowering effects associated with the H− X447 haplotype on fasting and postprandial TGs are not large, they are consistent with the lowering effect observed on MI risk throughout Europe.

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.