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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.

Activation of type-1 dopamine receptors ( DRD1 ) reduces renal sodium reabsorption. In a family-based random sample of 611 untreated whites (women, 45.0%; mean age, 38.6 years), we measured blood pressure (BP). We used the endogenous lithium clearance to assess fractional sodium excretion (FE Na ) and proximal (RNa prox ) and distal (RNa dist ) tubular sodium reabsorption. We investigated multivariate-adjusted associations with the DRD1 promoter ( A − 48G , G − 94A , and C − 800T ) and GRK4 (Ala142Val ). The frequent DRD1 haplotypes were AGC (48.2%), GGT (34.4%), and AAC (14.3%). While standardizing to mean sodium excretion (8.7 mmol/h) and adjusting for covariates and relatedness, RNa dist was lower in DRD1 − 94GG homozygotes than − 94A allele carriers (effect size, −0.94%; P =0.005) with opposite findings for FE Na (+0.084%; P =0.014). AGC carriers (−0.88%; P =0.012) and AAC carriers (+1.00%; P =0.004) had different RNa dist compared to corresponding noncarriers. Furthermore, FE Na was lower in AAC carriers than in noncarriers (−0.082%; P =0.019). The family-based analyses identified a significant between-family component in the variance of the renal phenotypes associated with the DRD1 polymorphisms. Transmission of the DRD1 AGC haplotype was also associated with lower systolic (−3.54 mm Hg; P =0.016) and diastolic (−2.80 mm Hg; P =0.0064) BPs without significant between-family variance component. Plasma renin activity and urinary aldosterone excretion were not associated with DRD1 variation. The GRK4 Ala142Val polymorphism did not contribute to the phenotypes under study. In conclusion, renal sodium handling and BP were associated with genetic variation in the DRD1 promoter. The between-family variance component excluded population stratification for BP, but not for the renal phenotypes.

See related articles, pages 1109–1120. Cardiovascular disease (CVD), including coronary artery disease (CAD) with its fatal clinical outcome myocardial infarction (MI) and stroke, remain the leading cause of deaths in western and nonwestern countries.1,2 In the US, CVD claims more lives than cancer, chronic lower respiratory tract diseases and diabetes mellitus combined, accounting for ≈36% of all deaths in 2004 and incurring an estimated direct and indirect cost of ≈432 billion dollars in 2007.2 Stroke and MI are multifactorial traits with complex pathophysiology. Besides other established factors, family history of disease strongly and independently determines CVD risk.3 There is a nearly 5-fold difference in stroke risk between monocygotic and dizygotic twins,4 and with respect to race, stroke incidence is approximately twice as high in blacks compared to whites.5 Marenberg et al,6 in their study on ≈20 000 Swedish twins, demonstrated that the relative risk to die from MI, when the other twin already died from this disease, was twice as large for monozygotic compared to dizygotic twins. Celera Genomics7 and the International Human Genome Project consortium,8 by whole genome shotgun and clone-based sequencing strategies, respectively, proposed 2 versions of the human genome—the diploid genome offering more genetic information than previously anticipated.9 The availability of almost the entire human genome sequence allows for the immediate access to specific target sequences across the human genome. The number of different technologies to explore genetic sequences on a “genome-wide” scale and high-throughput genotyping methods is steadily increasing. Genome-wide approaches are essentially based on the fact that alleles at nearby loci often show strong linkage disequilibrium (LD). Human recombination events occur ubiquitously in short genomic hotspots (1 to 2 kb) every 100 to 200 kb, preferentially outside genes,10 leading to a breakdown of allelic …

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 (

Objectives— Apolipoprotein E (apoE) exerts antiatherogenic effects but precise mechanisms remain unclear. We here investigated the effect of apoE on intracellular signaling by interleukin-1β (IL-1β), a proinflammatory cytokine present in atherosclerotic lesions. Methods and Results— IL-1β-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-κB and activating protein-1 (AP-1) transactivation, suggesting that the interruption of IL-1β signaling occurs upstream of transcription factors. Studies in VSMCs overexpressing IL-1β signaling intermediates revealed that NF-κB 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-1β 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-1β 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-α or polyinosinic:polycytidylic acid. Conclusion— ApoE targets IRAK-1 activation and thereby interrupts IL-1β and IL-18 signaling in VSMCs. This antiinflammatory effect represents a novel antiatherogenic activity of apoE.

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.

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.

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.