Hypertension is a common disorder of multifactorial origin that constitutes a major risk factor for cardiovascular events such as stroke and myocardial infarction. Previous studies demonstrated an enhanced signal transduction via pertussis toxin-sensitive G proteins in lymphoblasts and fibroblasts from selected patients with essential hypertension. We have detected a novel polymorphism (C825T) in exon 10 of the gene encoding the beta3 subunit of heterotrimeric G proteins (GNB3). The T allele is associated with the occurrence of a splice variant, GNB3-s (encoding G beta3-s), in which the nucleotides 498-620 of exon 9 are deleted. This in-frame deletion causes the loss of 41 amino acids and one WD repeat domain of the G beta subunit. By western-blot analysis, G beta3-s appears to be predominantly expressed in cells from individuals carrying the T allele. Significant enhancement of stimulated GTPgammaS binding to Sf9 insect cells expressing G beta3-s together with G alpha(i)2 and G gamma5 indicates that this splice variant is biologically active. Genotype analysis of 427 normotensive and 426 hypertensive subjects suggests a significant association of the T allele with essential hypertension.

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Several rare mutations in the melanocortin-4 receptor gene (MC4R) predispose to obesity. For the most common missense variant V103I (rs2229616), however, the previously reported similar carrier frequencies in obese and nonobese individuals are in line with in vitro studies, which have not shown a functional implication of this variant. In the present study, we initially performed a transmission/disequilibrium test on 520 trios with obesity, and we observed a lower transmission rate of the I103 allele (P=.017), which was an unexpected finding. Therefore, we initiated two large case-control studies (N=2,334 and N=661) and combined the data with those from 12 published studies, for a total of 7,713 individuals. The resulting meta-analysis provides evidence for a negative association of the I103 allele with obesity (odds ratio 0.69; 95% confidence interval 0.50-0.96; P=.03), mainly comprising samples of European origin. Additional screening of four other ethnic groups showed comparable I103 carrier frequencies well below 10%. Genomic sequencing of the MC4R gene revealed three polymorphisms in the noncoding region that displayed strong linkage disequilibrium with V103I. In our functional in vitro assays, the variant was indistinguishable from the wild-type allele, as was the result in previous studies. This report on an SNP/haplotype that is negatively associated with obesity expands the successful application of meta-analysis of modest effects in common diseases to a variant with a carrier frequency well below 10%. The respective protective effect against obesity implies that variation in the MC4R gene entails both loss and gain of function.

Abstract-G i proteins mediate the intracellular effects of many vasoactive and proliferative stimuli. Recently such signaling was found to be enhanced in cultured cells of some hypertensive subjects. A polymorphism at position 825 (C3 T) of the G protein ␤3 subunit gene (GNB3) was strictly related to this phenotype. The aim of the present investigation was to test the association between this polymorphism and blood pressure and plasma renin levels in humans. A population-based sample (nϭ608) was analyzed by questionnaire and characterized for blood pressure; plasma renin, prorenin, and aldosterone levels; and G ␤3 C825T allele status. In individuals without antihypertensive medication (nϭ474; age range, 52 to 67 years), the polymorphism was mildly associated with diastolic blood pressure (CC: 88.6Ϯ0.3 mm Hg, nϭ218; versus CT: 90.1Ϯ0.7 mm Hg, nϭ209; versus TT: 91.8Ϯ1.7 mm Hg, nϭ47; Pϭ0.02 for trend) but not with systolic blood pressure. Furthermore, the 825T allele was also significantly associated with lower renin and prorenin levels, whereas the aldosterone to renin ratio was elevated in these subjects. Significant associations between the 825T allele and diastolic blood pressure, plasma renin, and prorenin levels (inverse), and the aldosterone to renin ratio persisted after adjustment for age, gender, body mass index, and systolic blood pressure. Finally, when the entire sample was considered and an adjustment was made for covariates, the presence of arterial hypertension and the use of antihypertensive medication were both 1.8-fold higher in the TT than in the CC genotype group (PϽ0.05 and Pϭ0.06, respectively). This observation, if replicated in further studies, suggests a molecular mechanism that unifies a higher diastolic blood pressure, a lower renin level, and an elevated aldosterone to renin ratio, ie, a combination of features frequently found in patients with arterial hypertension. 1 In particular, immortalized lymphoblasts and fibroblasts of highly selected hypertensive subjects were found to respond with enhanced G protein-mediated activation of intracellular effectors after stimulation of various receptors.2,3 These specific receptors had in common the ability to activate pertussis toxinsensitive, G i -type G proteins. 2,3 The finding that immortalized cells of hypertensive subjects displayed enhanced G proteinmediated signaling suggested a genetic alteration rather than a blood pressure effect as the underlying mechanism. Subsequently, the genes coding for the G protein subunits G i␣2 , G i␣3 , G ␤1 , and G ␤2 were screened for polymorphisms, but were found to be without alterations in affected hypertensive subjects.3 However, molecular analysis of G ␤3 revealed a point polymorphism C825T that is associated with a novel splice variant and an in-frame deletion of 41 amino acids from the wild-type protein. 4 Enhanced sensitivity to agonists that stimulate intracellular signaling via pertussis toxinsensitive G i proteins was tightly associated with this splice variant and expression of recombinant mu...

(GTPyS), to membrane G proteins were not different in NT and HT cell lines. However, PAF-and mastoparan-stimulated binding of GTPyS to G proteins, which was fully PTX-sensitive, was 2.5-fold higher in HT than NT cell lines. These data suggest an enhanced receptor-mediated activation of PTX-sensitive G proteins despite unchanged receptor and G protein expression. Thus, this study not only suggests that enhanced signal transduction and cell proliferation are abnormalities in a certain group of patients with essential hypertension but also explains these findings as a result of an enhanced G protein activation in this common disorder.

Background-Recent clinical trials have suggested that intensive versus standard lipid-lowering therapy provides for additional benefit. Electron-beam computed tomography provides the opportunity to quantify the progression of coronary artery calcification (CAC) in serial measurements. Methods and Results-In a multicenter, randomized, double-blind trial, 471 patients (age 61Ϯ8 years) who had no history of coronary artery disease and no evidence of high-grade coronary stenoses (Ͼ50% diameter reduction) were randomized if they had Ն2 cardiovascular risk factors and moderate calcified coronary atherosclerosis as evidenced by a CAC score Ն30. Patients were assigned to receive 80 mg or 10 mg of atorvastatin per day over 12 months. Progression of CAC volume scores could be analyzed in 366 patients. After pretreatment with 10 mg of atorvastatin for 4 weeks, 12 months of study medication reduced LDL cholesterol from 106Ϯ22 to 87Ϯ33 mg/dL in the group randomized to receive 80 mg of atorvastatin (PϽ0.001), whereas levels remained stable in the group randomized to receive 10 mg (108Ϯ23 at baseline, 109Ϯ28 mg/dL at the end of the study, PϭNS). The mean progression of CAC volume scores, corrected for the baseline CAC volume score, was 27% (95% CI 20.8% to 33.1%) in the 80-mg atorvastatin group and 25% (95% CI 19.1% to 30.8%) in the 10-mg atorvastatin group (Pϭ0.65). CAC progression showed no relationship with on-treatment LDL cholesterol levels. Conclusions-We did not observe a relationship between on-treatment LDL cholesterol levels and the progression of calcified coronary atherosclerosis. Over a period of 12 months, intensive atorvastatin therapy was unable to attenuate CAC progression compared with standard atorvastatin therapy. The possibility remains that the time window was too short to demonstrate an effect. (Circulation. 2006;113:427-437.)

The polymerase chain reaction (PCR) technique has become an indispensable method in molecular research. However, PCR-amplification of GC-rich templates is often hampered by the formation of secondary structures like hairpins and higher melting temperatures. We present a novel method termed 'Slowdown PCR', which allows the successful PCR-amplification of extremely GC-rich (>83%) DNA targets. The protocol relies on the addition of 7-deaza-2'-deoxyguanosine, a dGTP analog to the PCR mixture and a novel standardized cycling protocol with varying temperatures. The latter consists of a generally lowered ramp rate of 2.5 degrees C s(-1) and a low cooling rate of 1.5 degrees C s(-1) for reaching an annealing temperature and is run for 48 cycles. We established this protocol as a versatile method not only for amplification of extremely GC-rich regions, but also for routine DNA diagnostics and pharmacogenetics for templates with different annealing temperatures. The protocol takes 5 h to complete.