Polymorphic variation in the 11beta-hydroxylase gene associates with reduced 11-hydroxylase efficiency.

The -344 C/T and intron 2 conversion variants in the CYP11B2 gene, encoding aldosterone synthase, have been associated with markers of impaired 11beta-hydroxylase activity. We hypothesize that this association is because of variations in the adjacent 11beta-hydroxylase gene (CYP11B1) and arises through linkage disequilibrium between CYP11B1 and CYP11B2. The pattern of variation across the entire CYP11B locus was determined by sequencing 26 normotensive subjects stratified by and homozygous for the -344 and intron conversion variants. Eighty-three variants associated with -344 and intron conversion were identified. Haplotype analysis revealed 4 common haplotypes, accounting for 68% of chromosomes, confirming strong linkage disequilibrium across the region. Two novel CYP11B1 polymorphisms upstream of the coding region (-1889 G/T and -1859 A/G) were identified as contributing to the common haplotypes. Given the potential for such mutations to affect transcriptional regulation of CYP11B1, these were analyzed further. A total of 512 hypertensive subjects from the British Genetics of Hypertension Study population were genotyped for these polymorphisms. A significant association was identified between the -1889 polymorphism and urinary tetrahydrodeoxycortisol/total cortisol metabolite ratio, indicating reduced 11beta-hydroxylase efficiency. A similar pattern was observed for the -1859 polymorphism, but this did not achieve statistical significance. Functional studies in vitro using luciferase reporter gene constructs show that these polymorphisms significantly alter the transcriptional response of CYP11B1 to stimulation by adrenocorticotropic hormone or forskolin. This study strongly suggests that the impaired 11beta-hydroxylase efficiency associated previously with the CYP11B2 -344 and intron conversion variants is because of linkage with these newly identified polymorphisms in CYP11B1.

Corcoran Lecture. Cardiovascular genomics and oxidative stress.

The majority of modifiable cardiovascular risk factors are complex, polygenic, or at least oligogenic traits, with genetic and environmental determinants playing important roles in disease risk and its phenotypic expression. The Human Genome Project and subsequent mouse and rat genome data have provided powerful tools to commence the dissection of genetic determinants of hypertension and other cardiovascular risk factors. Despite several new methodologies such as genome-wide scans, genome-wide gene expression profiling, and proteomic screens, it is fair to say that the progress of genetic studies designed as nonhypothesis driven has been relatively slow. On the other hand, several interesting candidate pathways have been identified, where investigators allowed for hypothesis-driven functional studies. One example of such pathway is vascular oxidative stress with its extensive network of genes and proteins, many with proven contributions to cardiovascular disease. Therefore, in parallel to genome-wide or proteome-wide studies, it will be constructive to pursue "pathwayomics" defined here as functional studies of a candidate pathway for disease pathogenesis.

Development of efficient viral vectors selective for vascular smooth muscle cells.

The vascular smooth muscle cell (SMC) is integral to the pathogenesis of neointimal formation associated with late vein graft failure, in-stent restenosis, and transplant arteriopathy. Viral vectors transduce SMC with low efficiency and hence, there is a need for improvement. We aimed to enhance the efficiency and selectivity of gene delivery to human SMC. Targeting ligands were identified using phage display on primary human saphenous vein SMC with linear and cyclic libraries. Two linear peptides, EYHHYNK (EYH) and GETRAPL (GET), were incorporated into the HI loop of adenovirus (Ad) fibers and the capsid protein of adeno-associated virus-2 (AAV-2). Exposure of human venous SMC to EYH-modified (but not the GET-modified) Ad vector resulted in a significant increase in transgene expression levels at short, clinically relevant exposure times. Similarly, the EYH-modified AAV vector resulted in enhanced gene transfer to human venous SMC but not endothelial cells in a time- and dose-dependent manner. The EYH-modified AAV vector also enhanced (up to 70-fold) gene delivery to primary human arterial SMC. Hence, incorporation of EYH into Ad and AAV capsids resulted in a significant and selective enhancement in transduction of SMC and has implications for improving local gene delivery to the vasculature.

Essential hypertension and beta2-adrenergic receptor gene: linkage and association analysis.

A region on human chromosome 5 (5q31.1-qter) contains several genes that encode important blood pressure regulators and thus is a good candidate for analysis of linkage and association with hypertension. We recruited 638 individuals from 212 Polish pedigrees with clustering of essential hypertension. These subjects were genotyped for 11 microsatellite markers that span this region to test for linkage to essential hypertension and systolic and diastolic blood pressures. The segment of this region of approximately 7 cM delineated by D5S1480 and D5S500 markers was linked to blood pressures in multipoint analysis. In 2-point analysis, D5S1480--the marker in close proximity to beta2-adrenergic receptor gene--reached the maximal linkage to essential hypertension and adjusted systolic and diastolic blood pressures, implicating this gene as a positional candidate for further association studies. Arg16Gly, Gln27Glu, and Thr164Ile--3 functional single nucleotide polymorphisms within the beta2-adrenergic receptor gene--were tested for association with essential hypertension. None of these polymorphisms showed a significant association with essential hypertension, separately or in the haplotype analysis. This study provided evidence of linkage of 5q31.1-5qter region to essential hypertension in the European population. Moreover, it implicated the chromosomal segment in close proximity to D5S1480 and D5S500. The detailed analysis of 3 single nucleotide polymorphisms does not support the role of the beta2-adrenergic receptor gene as a major causative gene for the detected linkage.