A follow-up study of a genome-wide association scan identifies a susceptibility locus for venous thrombosis on chromosome 6p24.1.

To identify genetic susceptibility factors conferring increased risk of venous thrombosis (VT), we conducted a multistage study, following results of a previously published GWAS that failed to detect loci for developing VT. Using a collection of 5862 cases with VT and 7112 healthy controls, we identified the HIVEP1 locus on chromosome 6p24.1 as a susceptibility locus for VT. Indeed, the HIVEP1 rs169713C allele was associated with an increased risk for VT, with an odds ratio of 1.20 (95% confidence interval 1.13-1.27, p = 2.86 x 10(-9)). HIVEP1 codes for a protein that participates in the transcriptional regulation of inflammatory target genes by binding specific DNA sequences in their promoter and enhancer regions. The current results provide the identification of a locus involved in VT susceptibility that lies outside the traditional coagulation/fibrinolysis pathway.

Molecular genetic analysis of a human insulin-like growth factor 1 promoter P1 variation.

Insulin-like growth factor 1 (IGF1) exerts important endocrine and paracrine functions in the cardiovascular system. We identified the common variant -1411C>T in the IGF1 upstream promoter P1, located within several overlapping transcription factor binding sites. Using transient transfection assays, we identified this site as a functional enhancer. The T allele-carrying enhancer, compared with the C allelic portion, exerts significantly reduced or even abrogated activity, respectively, in SaOs-2 and HepG2 (all P<0.0001) as well as in differentiated THP-1 macrophages. Electrophoretic mobility shift assay and subsequent supershift experiments in HepG2 identified c-Jun as the binding partner exclusively to the T allele, whereas CCAAT/enhancer-binding protein delta and interferon consensus site-binding protein/interferon-regulating factor 8 interacted only with the C allelic promoter portion. Furthermore, genotyping in a case-control study for essential hypertension (n=745 hypertensive patients; n=769 normotensive control subjects) for this variant revealed an odds ratio for hypertension of 0.73 (95% confidence interval 0.58-0.91, P=0.006) associated with the T allele, and normotensive subjects carrying the protective T allele displayed a significant decrease in diastolic (P=0.036) and systolic (P=0.024) blood pressure levels. We here report detection of a functional enhancer module in the upstream IGF1 promoter region, which might play a key role in local IGF1 bioavailability. Whether -1411C>T is also associated with other IGF1-related disease phenotypes should be evaluated further in population studies.

Identification and functional analyses of molecular haplotypes of the human osteoprotegerin gene promoter.

OBJECTIVE: Osteoprotegerin (OPG) has been reported to be involved in the development of atherosclerotic disease, and OPG gene variation has been associated with plasma OPG levels and different cardiovascular disease phenotypes. However, the genetic architecture of the OPG promoter and its transcriptional regulation are poorly characterized. METHODS AND RESULTS: We identified 1008 bp of the OPG 5'-flanking region to be sufficiently transcriptionally active in osteosarcoma cell lines and generated serial promoter deletion constructs. Individual subcloning revealed the existence of 3 molecular haplotypes (MolHaps): [T(-960)-A(-946)-G(-900)-T(-864); MolHap1, wild type], [T(-960)-G(-946)-G(-900)-T(-864); MolHap2], [C(-960)-G(-946)-A(-900)-G(-864); MolHap4]. Compared to MolHap1, transcriptional activities of MolHaps 2 and 4 were significantly reduced (P=0.0018). Whereas introduction of the -159C allele reduced transcriptional activities of the full-length constructs (P=0.0014), it significantly increased activities of the deletion constructs (P=0.0005). Electrophoretic mobility shift, competition, and chromatin immunoprecipitation assays revealed specific DNA:protein interactions for the MolHaps with Sp1 and NF-1, and identified Egr1 interacting exclusively with the -159T allele. CONCLUSIONS: We propose new structural and transcriptional features within the OPG promoter region and identified MolHaps being differentially transcriptionally active and allele-dependently interacting with a proximal polymorphic site.

SAH gene variants revisited in the European Project On Genes in Hypertension.

OBJECTIVE: Previous studies found significant association of hypertension and hypertension-related phenotypes with genetic variation in SAH (Spontaneously hypertensive rat-clone A-Hypertension-associated). We sought independent confirmation of these findings in the European Project On Genes in Hypertension. METHODS AND RESULTS: We randomly recruited 2603 relatives from 560 families and 31 unrelated subjects from six European populations (mean age 38.8 +/- 15.7 years; 52.1% women). We measured systolic/diastolic blood pressure (mean, 122.4/76.6 mmHg), body mass index (24.9 kg/m2), triceps skinfold (1.7 cm), waist-to-hip ratio (0.83 units), serum total and high-density lipoprotein (HDL) cholesterol (5.14 and 1.33 mmol/l), serum triglycerides (1.95 mmol/l) and blood glucose (4.90 mmol/l). We genotyped the G-1606A and -962del/ins polymorphisms. In all subjects, the allele frequencies were 11.8 and 29.5% for -1606A and -962del, respectively. Lewontin's D' was 0.97 (P < 0.0001). Haplotype frequencies were 58.8% for -1606G plus -962ins, 29.5% for -1606G plus -962del, and 11.7% for -1606A plus -962ins. Both before and after adjustment for covariates, none of the phenotype-genotype associations approached statistical significance. Our study had 80% power to detect on two-sided tests (P = 0.05), effect sizes of 1.8/1.3 mmHg for systolic/diastolic blood pressure, 0.52 kg/m2 for body mass index, 0.01 units for the waist-to-hip ratio, 0.96 mm for the triceps skinfold, 0.13 and 0.05 mmol/l for total and HDL cholesterol, 0.18 mmol/l for serum triglycerides, and 0.11 mmol/l for blood glucose. The family-based analyses did not reveal population stratification (P > or = 0.67). CONCLUSION: The evidence supporting an association of hypertension or hypertension-related phenotypes with the SAH gene remains equivocal in human studies.

Apolipoprotein E interrupts interleukin-1beta signaling in vascular smooth muscle cells.

OBJECTIVES: Apolipoprotein E (apoE) exerts antiatherogenic effects but precise mechanisms remain unclear. We here investigated the effect of apoE on intracellular signaling by interleukin-1beta (IL-1beta), a proinflammatory cytokine present in atherosclerotic lesions. METHODS AND RESULTS: IL-1beta-induced expression and activation of inducible nitric oxide synthase and cyclooxygenase-2 were inhibited by apoE in vascular smooth muscle cells (VSMCs). These inhibitory effects were linked to the suppression of both NF-kappaB and activating protein-1 (AP-1) transactivation, suggesting that the interruption of IL-1beta signaling occurs upstream of transcription factors. Studies in VSMCs overexpressing IL-1beta signaling intermediates revealed that NF-kappaB transactivation was inhibited by apoE in MyD88- and IRAK1- but not in TRAF6-transfected cells. Furthermore, apoE prevented IRAK1 phosphorylation and IRAK1-TRAF6 but not MyD88-IRAK1 complex formation. Inhibitory effects of apoE on IL-1beta signaling were abolished after silencing LDL receptor-related protein-1 (LRP1) expression with siRNA. In addition, inhibitors of adenylyl cyclase and protein kinase A (PKA) restored IL-1beta signaling in apoE-treated VSMCs, whereas apoE stimulated PKA activity. ApoE inhibited VSMC activation in response to IL-18 but not to tumor necrosis factor-alpha or polyinosinic:polycytidylic acid. CONCLUSION: ApoE targets IRAK-1 activation and thereby interrupts IL-1beta and IL-18 signaling in VSMCs. This antiinflammatory effect represents a novel antiatherogenic activity of apoE.

Polymorphisms in 33 inflammatory genes and risk of myocardial infarction--a system genetics approach.

The hypothesis of a causal link between inflammation and atherosclerosis would be strengthened if variants of inflammatory genes were associated with disease. Polymorphisms of 33 genes encoding inflammatory molecules were tested for association with myocardial infarction (MI). Patients with MI and a parental history of MI (n = 312) and controls from the UK (n = 317) were genotyped for 162 polymorphisms. Thirteen polymorphisms were associated with MI (P values ranging from 0.003 to 0.041). For three genes, ITGB1, SELP, and TNFRSF1B haplotype frequencies differed between patients and controls (P values < 0.01). We further assessed the simultaneous contribution of all polymorphisms and relevant covariates to MI using a two-step strategy of data mining relying on Random Forest and DICE algorithms. In a replication study involving two independent samples from the UK (n = 649) and France (n = 706), one interaction between the ITGA4/R898Q polymorphism and current smoking status was replicated. This study illustrates a strategy for assessing the joint effect of a large number of polymorphisms on a phenotype that may provide information that single locus or single gene analysis may fail to uncover. Overall, there was weak evidence for an implication of inflammatory polymorphisms on susceptibility to MI.

-391 C to G substitution in the regulator of G-protein signalling-2 promoter increases susceptibility to the metabolic syndrome in white European men: consistency between molecular and epidemiological studies.

BACKGROUND: The regulator of G-protein signalling-2 (RGS2) is a key factor in adipogenesis. We hypothesized that the metabolic syndrome, of which obesity is an important component, might be related to genetic variation in RGS2. METHODS AND RESULTS: We screened the human RGS2 gene. We tested the functionality of a common genetic variant in vitro, ex vivo, and in epidemiological study involving six European populations. The C to G substitution at position -391 in the RGS2 promoter was associated with enhanced RGS2 expression in vitro in transfected 3T3-L1 adipocytes and Chinese hamster cells and ex vivo in adipocytes from male, but not female, volunteers. In 2732 relatives from 512 families and 348 unrelated individuals, randomly recruited from six European populations, the prevalence of GG homozygosity was 54.1%. The metabolic syndrome score, a composite of six continuous traits making up this clinical entity, was 0.27 standardized units higher (P < 0.001) in 795 GG homozygous men compared with 683 men carrying the C allele. Transmission of the -391 G allele to male offspring was associated with a 0.20 unit increase in the score (P=0.039). These epidemiological relations were not significant in 1602 women. CONCLUSIONS: The C to G substitution at position -391 in the RGS2 promoter increases RGS2 expression in adipocytes and is associated with the metabolic syndrome in white European men. Further experimental and clinical research should establish whether this common polymorphism might be a target for preventive or therapeutic intervention.

SAH gene variants are associated with obesity-related hypertension in Caucasians: the PEGASE Study.

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.

The G-231A polymorphism in the endothelin-A receptor gene is associated with lower aortic pressure in patients with dilated cardiomyopathy.

BACKGROUND: The endothelin system (ES) plays an important role in blood pressure (BP) regulation and also in the pathophysiology of idiopathic dilated cardiomyopathy (DCM). Recently, we demonstrated that a genetic polymorphism in the endothelin A (ET(A)) receptor gene was associated with survival in DCM patients. The aim of this study was to determine whether polymorphisms in the ET(A) receptor gene might be associated with the severity of DCM. METHODS: One hundred twenty-four consecutively recruited unrelated patients with DCM, who underwent a detailed phenotyping protocol, were genotyped for the ET(A) receptor G-231A polymorphism using a hybridization technique with allele-specific oligonucleotides. RESULTS: The exon 1 G-231A polymorphism of the ET(A) receptor gene, upstream of the translation start site, was significantly associated with directly measured intra-aortic pressure in that -231A allele carriers had significantly lower systolic (P = .0043), as well as mean (P = .0016) and diastolic (P = .0041) aortic pressure compared to noncarriers. The association of ET(A) G-231A with aortic pressure was independent from other factors such as prior medication, left ventricular end-diastolic diameter, age, gender, and New York Heart Association (NYHA) functional classification. However, no such association was seen for cuff BP and survival rates were not significantly different between -231A allele carriers and -231G homozygotes (log rank test, P = .66). No significant association with any other parameter investigated in the present study could be observed, even when men and women were analyzed separately. CONCLUSIONS: Our results suggest an association of genetic variation in the ET(A) receptor gene with aortic pressure in patients with DCM.

Cytokine polymorphisms associated with carotid intima-media thickness in stroke patients.

BACKGROUND AND PURPOSE: Carotid intima-media thickness (IMT) reflects generalized atherosclerosis and is predictive of future vascular events. Evidence exists that carotid IMT is heritable, and genetic studies can provide clues in the pathogenesis of atherosclerosis. METHODS: We recruited 470 white ischemic stroke patients, measured common carotid artery (CCA) IMT, and analyzed 54 polymorphisms with suspected roles in atherosclerosis. RESULTS: Among the polymorphisms tested, the angiotensin-converting enzyme insertion/deletion, osteopontin (OPN) T-443C, monocyte chemoattractant protein-1 (MCP-1) G-927C, and MCP-1 A-2578G polymorphisms were associated with CCA-IMT in age-gender-adjusted analysis. In multivariate analysis, the association between the OPN and MCP-1 polymorphisms remained significant. The OPN-443C allele was associated with increased IMT in the dominant model (0.053 mm for the TC and CC genotypes; P=0.001). The MCP-1-927C allele was associated with increased IMT in the additive model (0.040 mm for each C allele; P=0.001), and the MCP-1-2578 G allele was associated with decreased IMT in the recessive model (0.088 mm for the GG genotype; P=0.002). CONCLUSIONS: The OPN and MCP-1 genes, coding for 2 cytokines with known roles in atherosclerosis, may contribute to increased carotid IMT and warrant further study.

Sodium excretion as a modulator of genetic associations with cardiovascular phenotypes in the European Project on Genes in Hypertension.

Hypertension is a chronic age-related disorder, affecting nearly 20% of all adult Europeans. This disease entails debilitating cardiovascular complications and is the leading cause for drug prescriptions in Europeans older than 50 years. Intensive research over the past two decades has so far failed to identify common genetic polymorphisms with a major impact on blood pressure or associated cardiovascular phenotypes, suggesting that multiple genes each with a minor impact, along with gene-gene and gene-environment interactions, play a role. The European Project on Genes in Hypertension (EPOGH) is a large-scale, family-based study in which participants from seven different populations were phenotyped and genotyped according to standardized procedures. This review article summarizes the initial 5-year findings and puts these observations into perspective against other published studies. The EPOGH demonstrated that phenotype-genotype relations strongly depend on host factors such as gender and lifestyle, in particular salt intake as reflected by the 24-h urinary excretion of sodium. The EPOGH therefore highlights the concept that phenotype-genotype relations can only be studied within a defined ecogenetic context.

Left ventricular mass in relation to genetic variation in angiotensin II receptors, renin system genes, and sodium excretion.

BACKGROUND: In the European Project On Genes in Hypertension (EPOGH), we investigated in 3 populations to what extent left ventricular mass (LVM) was associated with genetic variation in the angiotensin II receptors type 1 (AGTR1 A1166C) and type 2 (AGTR2 G1675A) while accounting for possible gene-gene interactions with the angiotensin-converting enzyme (ACE D/I) and angiotensinogen (AGT -532C/T) polymorphisms. METHODS AND RESULTS: We randomly recruited 221 nuclear families (384 parents, 431 offspring) in Cracow (Poland), Novosibirsk (Russia), and Mirano (Italy). Echocardiographic LVM was indexed to body surface area, adjusted for covariates, and subjected to multivariate analyses using generalized estimating equations and quantitative transmission disequilibrium tests in a population-based and family-based approach, respectively. For AGTR1 and AGTR2, there was no heterogeneity in the phenotype-genotype relations across populations. LVM index was unrelated to the AGTR1 A1166C polymorphism. In men, in the population- and family-based analyses, the allelic effects of the AGTR2 polymorphism on LVM index differed (P=0.01) according to sodium excretion. In women, this gene-environment interaction did not reach statistical significance. In untreated men, LVM index (4.2 g/m2 per 100 mmol) and left ventricular internal diameter (0.73 mm/100 mmol) increased (P<0.02) with higher sodium excretion in the presence of the G allele with an opposite tendency in A allele carriers. The ACE D/I polymorphism, together with the ACE genotype-by-sodium interaction term, significantly and independently improved the models relating LVM index to the AGTR2 polymorphism and the AGTR2 genotype-by-sodium interaction. CONCLUSIONS: The present findings support the hypothesis that in men the AGTR2 G1675A and the ACE D/I polymorphisms independently influence LVM and that salt intake modulates these genetic effects.

Angiotensinogen promoter haplotypes are associated with blood pressure in untreated hypertensives.

BACKGROUND: The polymorphic angiotensinogen (AGT) gene is one of the most promising candidates for blood pressure (BP) regulation and essential hypertension. OBJECTIVES: To investigate whether AGT haplotype analysis adds significant information compared to single polymorphism analysis with respect to different BP phenotypes in an untreated hypertensive sample. METHODS: Two hundred and twelve untreated hypertensive subjects of Caucasian origin were genotyped for the AGT polymorphisms C-532T, A-20C, C-18T, and G-6A. RESULTS: In single variant analyses, untreated hypertensives, carrying the AGT -532T or -6A alleles had significantly higher systolic blood pressure (SBP) and diastolic blood pressure (DBP), as well as ambulatory BP values compared to respective non-carriers. In haplotype-based analyses, combining all four AGT promoter variants, we demonstrate that AGT haplotypes containing different allele combinations at positions -532 and -6 were significantly associated with different BP values: (1) -532T and -6A with higher, (2) -532C and -6G with lower, (3) -532C and -6A with intermediate BP values. Since the result for the -532C/-20A/-18C/-6G haplotype was due to differences between non-carriers and carriers of this haplotype on both chromosomes, a recessive inheritance model for BP effects could be assumed. CONCLUSIONS: Our results designate the C-532T and G-6A as the best candidates for functional studies on the AGT gene. Haplotype-based analyses should greatly aid in the dissection of the genetic basis of complex traits, such as BP regulation and hypertension.

Hypertension in mice lacking the CXCR3 chemokine receptor.

The CXC chemokine receptor 3 (CXCR3) has been linked to autoimmune and inflammatory disease, allograft rejection, and ischemic nephropathy. CXCR3 is expressed on endothelial and smooth muscle cells. Although a recent study posited that antagonizing of CXCR3 function may reduce atherosclerosis, the role of CXCR3 in controlling physiological vascular functions remains unclear. This study demonstrates that disruption of CXCR3 leads to elevated mean arterial pressures in anesthetized and conscious mice, respectively. Stimulation of isolated resistance vessels with various vasoconstrictors showed increased contractibility in CXCR3-/- mice in response to angiotensin II (ANG II) and a decreased vasodilatation in response to acetylcholine (ACh). The increased contractibility was related to higher ANG II type 1 receptor (AT1R) expression, whereas the decreased vasodilatation was related to lower M3-ACh receptor expression in the mesenteric arteries of CXCR3-/- mice compared with wild-type mice. The vasodilatatory response to ACh could be antagonized by the nonselective ACh receptor antagonist atropine and the selective M3 receptor antagonist 4-DAMP, but not by M1, M2, and M4 receptor antagonists. Additionally, EMSA studies revealed that transcription factors SP-1 and EGR-1 interact as a complex with the murine AT1R promoter region. Furthermore, we could show increased expression of SP-1 in CXCR3-/- mice indicating an imbalanced SP-1 and EGR-1 complex formation which causes increased AT1R expression and hypertension. The data indicate that CXCR3 receptor is important in vascular contractility and hypertension, possibly through upregulated AT1R expression.

Osteopontin gene variation and cardio/cerebrovascular disease phenotypes.

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.

Left ventricular structure in relation to the human SAH gene in the European Project on Genes in Hypertension.

Earlier studies showed association of the human SAH (Spontaneously hypertensive rat-clone A-Hypertension associated) gene with hypertension and obesity. Left ventricular mass index (LVMI) increases with blood pressure and body mass index. In a family-based population study (54.5% women; mean age, 43.1 years), we measured LVMI, mean wall thickness (MWT) and the left ventricular internal diameter (LVID) at end-diastole in 699 non-Slavic and 493 Slavic participants. In multivariable-adjusted analyses, we investigated phenotype-genotype associations (SAH G-1606A and -962ins/del polymorphisms), while accounting for confounders and relatedness. Non-Slavic -1606GG homozygotes had a slightly greater LVID than -1606A allele carriers (48.6 vs. 48.0 mm; P=0.08). However, the between-family component of the variance in LVID was significant (P=0.005), suggesting that population stratification might explain the latter finding. Non-Slavic -962del carriers had higher LVMI (91.1 vs. 88.5 g m(-2); P=0.03) and MWT (9.61 vs. 9.44 mm; P=0.03) than -962ins homozygotes. Transmission of the -962del to non-Slavic offspring was also associated with higher MWT (P=0.03). In Slavic participants, in the absence of population stratification (P>or=0.69), -1606GG homozygotes had lower LVMI (96.5 vs. 102.3 g m(-2); P=0.004) and lower MWT (10.1 vs. 10.5 mm; P=0.003) than -1606A carriers. Sensitivity analyses showed that the latter associations were confined to founders. Transmission of the -962del allele to Slavic offspring was associated with lower MWT (P=0.007). In conclusion, LVMI and MWT, two phenotypes that are jointly influenced by blood pressure and obesity, might be related to variation in the human SAH gene.

Cardiovascular risk factors and mortality in patients with coronary heart disease.

A priority in preventive cardiology is to reduce the number of recurrent events and to prolong survival in patients with established coronary heart disease (CHD). Aim of the present study was to examine risk factors for long-term mortality in CHD patients who entered routine secondary prevention after a coronary event or intervention. Such patients, from the EUROASPIRE (EUROpean Action on Secondary Prevention through Intervention to Reduce Events) I and II studies in the region of Münster, Germany, were followed over a mean period of 8.0 years up to the end of 2005. Patients were up to 70 years of age at baseline when they were interviewed and examined using standardised methods. Baseline examination was carried out at least 6 months and at a mean of 19.5 months after the coronary event or procedure. In 367 patients from EUROASPIRE I and 380 patients from EUROASPIRE II, a total of 125 deaths (16.7%) occurred during follow-up. Multivariate analyses, using Cox proportional hazards models, established diabetes mellitus and smoking as predictors for all-cause mortality with estimated hazard rate ratios (HRRs) of 2.24 (95% confidence interval (CI): 1.43-3.49) and 1.95 (95% CI: 1.23-3.10), respectively. Significant associations were found between diabetes mellitus and cardiovascular (HRR 2.36; 95% CI: 1.31-4.24) as well as CHD mortality (HRR 2.40; 95% CI: 1.25-4.59). Systolic blood pressure was significantly associated with increased cerebrovascular disease mortality (HRR 1.04; 95% CI: 1.01 and 1.08 for 1 mmHg increase). In conclusion, long-term mortality in coronary patients from routine secondary prevention is substantial. Diabetes mellitus and smoking represent key issues in patients with established CHD.

Characterization and functional analyses of the human G protein-coupled receptor kinase 4 gene promoter.

The G protein-coupled receptor kinase 4 is involved in renal sodium handling and blood pressure regulation. Missense variants have already been tested functionally and are associated with hypertension, but no data on promoter analyses are yet available. We scanned 94 hypertensive white subjects for genetic variation and performed promoter reporter gene analyses in HEK293T, COS7, and SaOs-2 cells. Transient transfections with various full lengths and wild-type deletion constructs revealed that 1851 bp of the flanking region and 275 bp of the 5'-untranslated region were sufficient for transcriptional activities and composed a powerful cis-active element in the distal 293 bp. The -1702T and +2T alleles resulted in drastic general reductions of promoter function, whereas an activity increasing effect of +268C was cell type specific. Electrophoretic mobility-shift assay, supershift, and cotransfection analyses of transcription factor binding sites predicted in silico (Alibaba2.1/Transfac7) resulted in allele-specific binding patterns of nuclear proteins and identified the participation of CCAAT/enhancer-binding protein transcription factor family members. The G protein-coupled receptor kinase 4 core promoter resides in the first 1851 bp upstream of its transcription start site. The 4 identified genetic variants within this region exert allele-specific impact on both cell type- and stimulation-dependent transcription and may affect the expression balance of renal G protein-coupled receptor kinase 4.

Functional and structural profiling of the human thrombopoietin gene promoter.

Human thrombopoietin (TPO) is involved in cardiovascular disease as it regulates megakaryocyte development and enhances platelet adhesion/aggregation. The THPO promoter structure is still controversial. By reverse transcription-PCR, we confirm that THPO transcription is cell line-dependently initiated at two alternative promoters, which we newly designated P1a and P1. We subsequently electrophoretically scanned and resequenced these portions in 95 and 57 patients with cardiovascular disease, respectively, and identified seven variants (-1450/del58bp, C-920T [rs2855306], A-622G, C-413T [rs885838], C+5A, G+115A, and C+135T). After subcloning of 1032 bp of THPO P1 in pGL3-basic vector, five molecular haplotypes (MolHaps1-5) were observed: [A(-622)-C(-413)-C(+5)-G(+115); wild type (wt)], [A(-622)-T(-413)-C(+5)-G(+115)], [G(-622)-T(-413)-C(+5)-G(+115)], [A(-622)-C(-413)-A(+5)-G(+115)], [A(-622)-C(-413)-C(+5)-A(+115)], and analyzed in reporter gene assays in HEK293T and HepG2 cells. MolHaps 2, 4, and 5 were significantly more active than wt (all p values < or =0.01) in HEK293T cells, MolHap3 exerted a substantial loss of promoter activity (p < 0.0001 in HEK293T and p < 0.01 in HepG2, compared with wt). Electrophoretic mobility shift assays revealed that A-622G and C-413T individually differed from MolHaps in their DNA-protein interaction patterns. Supershift and chromatin immunoprecipitation assays identified CCAAT/enhancer-binding protein delta as the binding protein exclusively for the -622A allelic portion.