Stefan-Martin Brand-Herrmann

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.

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.

Blood Pressure and Renal Sodium Handling in Relation to Genetic Variation in the DRD1 Promoter and GRK4

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 (FENa) and proximal (RNaprox) and distal (RNadist) 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, RNadist was lower in DRD1 −94GG homozygotes than −94A allele carriers (effect size, −0.94%; P=0.005) with opposite findings for FENa (+0.084%; P=0.014). AGC carriers (−0.88%; P=0.012) and AAC carriers (+1.00%; P=0.004) had different RNadist compared to corresponding noncarriers. Furthermore, FENa 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.

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.

Alcohol intake modulates the genetic association between HDL cholesterol and the PPARgamma2 Pro12Ala polymorphism.

The peroxisome proliferator-activated receptor γ (PPARγ) Pro12Ala polymorphism affects plasma lipids, but to what extent alcohol intake interferes with this association remains unknown. We randomly recruited 251 nuclear families (433 parents and 493 offspring) in the framework of the European Project on Genes in Hypertension study and genotyped 926 participants in whom all serum lipid variables and information on alcohol consumption were available for PPARγ2 Pro12Ala. Genotype-phenotype relations were assessed using generalized estimating equations (GEE) and a quantitative transmission disequilibrium test (QTDT). The Ala12 allele was more frequent in Novosibirsk (0.17) than in Cracow (0.12) and Mirano (0.11) (P < 0.01). Using GEE (P = 0.03) or QTDT (P = 0.007), Italian offspring carrying the Ala12 allele had higher serum HDL cholesterol than noncarriers. HDL cholesterol levels were on average 0.086 mmol/l (P = 0.001) higher in drinkers than in nondrinkers. Compared with Pro12 homozygotes, Ala12 allele carriers consuming alcohol had higher serum total and HDL cholesterol, with the opposite trend occurring in nondrinkers. This genotype-alcohol interaction was independent of the type of alcoholic beverage and more pronounced in moderate than in heavy drinkers. We conclude that alcohol intake modulates the relation between the PPARγ2 Pro12Ala and HDL cholesterol level and that, therefore, the Pro12Ala polymorphism, pending confirmation of our findings, might affect cardiovascular prognosis.

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.

Apolipoprotein E Interrupts Interleukin-1β 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-1&bgr; (IL-1&bgr;), a proinflammatory cytokine present in atherosclerotic lesions. Methods and Results—IL-1&bgr;–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-&kgr;B and activating protein-1 (AP-1) transactivation, suggesting that the interruption of IL-1&bgr; signaling occurs upstream of transcription factors. Studies in VSMCs overexpressing IL-1&bgr; signaling intermediates revealed that NF-&kgr;B 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-1&bgr; 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-1&bgr; 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-&agr; or polyinosinic:polycytidylic acid. Conclusion—ApoE targets IRAK-1 activation and thereby interrupts IL-1&bgr; and IL-18 signaling in VSMCs. This antiinflammatory effect represents a novel antiatherogenic activity of apoE.

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, inSaOs‐2 and HepG2 (all p<0.0001) as well as in differentiatedTHP‐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 8 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, 7=0.006) associated with the T allele, and normotensive subjects carrying the protective T allele displayed a significant decrease in diastolic (7=0.036) and systolic (7=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.— Telgmann, R., Dordelmann, C., Brand, E., Nicaud, V., Hagedorn, C., Pavenstadt, H., Cambien, F., Tiret, L., Paul, M., Brand‐Herrmann, S.‐M. Molecular genetic analysis of a human insulin growth factor 1 promoter P1 variation. FASEBJ. 23, 1303–1313 (2009)

Arterial properties in relation to genetic variation in α-adducin and the renin–angiotensin system in a White population

Earlier studies have demonstrated the interaction between ADD1 and ACE in relation to arterial properties. We investigated whether arterial characteristics might also be related to interactions of ADD1 with other renin–angiotensin system genes. Using a family-based sampling frame, we randomly recruited 1064 Flemish subjects (mean age, 43.6 years; 50.4% women). By means of a wall-tracking ultrasound system, we measured the properties of the carotid, femoral and brachial arteries. In multivariate-adjusted analyses, we assessed the multiple gene effects of ADD1 (Gly460Trp), AGT (C–532T and G–6A) and AT1R (A1166C). In ADD1 Trp allele carriers, but not in ADD1 GlyGly homozygotes (P-value for interaction ⩽0.014), femoral cross-sectional compliance was significantly higher (0.74 vs 0.65 mm2 kPa−1; P=0.020) in carriers of the AT1R C allele than in AT1R AA homozygotes, with a similar trend for femoral distensibility (11.3 vs 10.2 × 10−3 kPa−1; P=0.055). These associations were independent of potential confounding factors, including age. Family-based analyses confirmed these results. Brachial diameter (4.35 vs 4.18 mm) and plasma renin activity (PRA) (0.23 vs 0.14 ng ml−1 h−1) were increased (P⩽0.005) in AGT CG haplotype homozygotes compared with non-carriers, whereas the opposite was true for brachial distensibility (12.4 vs 14.4 × 10−3 kPa−1; P=0.011). There was no interaction between AGT and any other gene in relation to the measured phenotypes. ADD1 and AT1R interactively determine the elastic properties of the femoral artery. There is a single-gene effect of the AGT promoter haplotypes on brachial properties and PRA.

Context-dependency of the relation between left ventricular mass and AGT gene variants

In the European Project on Genes in Hypertension (EPOGH), we investigated in three populations to what extent in a family-based study, left ventricular mass (LVM) was associated with the C−532T and G−6A polymorphisms in the angiotensinogen (AGT) gene. We randomly recruited 221 nuclear families (384 parents and 440 offspring) in Cracow (Poland), Novosibirsk (Russia), and Mirano (Italy). Echocardiographic LVM was indexed to body surface area, adjusted for covariables, and subjected to multivariate analyses, using generalized estimating equations and quantitative transmission disequilibrium tests in a population-based and family-based approach, respectively. We found significant differences between the two Slavic centres and Mirano in left ventricular mass index (LVMI) (94.9 vs 80.4 g/m2), sodium excretion (229 vs 186 mmol/day), and the prevalence of the AGT −6A (55.7 vs 40.6%) and −532T (16.8 vs 9.4%) alleles. In population-based as well as in family-based analyses, we observed positive associations of LVMI and mean wall thickness (MWT) with the −532T allele in Slavic, but not in Italian male offspring. Furthermore, in Slavic male offspring, LVMI and MWT were significantly higher in carriers of the −532T/−6A haplotype than in those with the −532C/−6G or −532C/−6A allele combinations. In women, LVMI was neither associated with single AGT gene variants nor with the haplotypes (0.19<P<0.98). In Slavic offspring carrying the AGT −532C/−6G or −532C/−6A haplotypes, LVMI significantly increased with higher sodium excretion (+3.5 g/m2/100 mmol; P=0.003), whereas such association was not present in −532T/−6A haplotype carriers (P-value for interaction 0.04). We found a positive association between LVMI and the AGT −532T allele due to increased MWT. This relation was observed in Slavic male offspring. It was therefore dependent on gender, age and ecogenetic context, and in addition it appeared to be modulated by the trophic effects of salt intake on LVM.