Abstract-The Ϫ344 C/T and intron 2 conversion variants in the CYP11B2 gene, encoding aldosterone synthase, have been associated with markers of impaired 11␤-hydroxylase activity. We hypothesize that this association is because of variations in the adjacent 11␤-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 Ϫ344 and intron conversion variants. Eighty-three variants associated with Ϫ344 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 (Ϫ1889 G/T and Ϫ1859 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 Ϫ1889 polymorphism and urinary tetrahydrodeoxycortisol/total cortisol metabolite ratio, indicating reduced 11␤-hydroxylase efficiency. A similar pattern was observed for the Ϫ1859 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 11␤-hydroxylase efficiency associated previously with the CYP11B2 Ϫ344 and intron conversion variants is because of linkage with these newly identified polymorphisms in CYP11B1. Key Words: hypertension Ⅲ aldosterone synthase Ⅲ 11␤-hydroxylase Ⅲ polymorphisms Ⅲ transcriptional regulation T he mineralocorticoid hormone aldosterone plays an important role in blood pressure homeostasis. In the Framingham population, plasma aldosterone levels in the top quartile of the normal distribution predicted subsequent rise in blood pressure and future development of hypertension. 1 Furthermore, Յ15% of unselected patients with hypertension have a raised aldosterone/renin ratio, suggesting that altered regulation of aldosterone synthesis or release may be a common factor in essential hypertension. [2][3][4][5][6] The late stages in aldosterone synthesis that occur in the zona glomerulosa of the adrenal cortex are key in the control of production of the hormone. The 3 terminal steps are a sequential 11␤-hydroxylation, 18-hydroxylation, and 18-oxidation of the precursor 11-deoxycorticosterone, all catalyzed by the enzyme aldosterone synthase. This enzyme is encoded by the CYP11B2 gene, the major regulator of which is angiotensin II. In the zona fasciculata, cortisol is synthesized by a similar pathway, but the final stage is an 1...

No variants were identified in the coding region of CYP11B1 that could account for hypertension and/or a raised ARR. However, this in vitro study identifies the importance of these affected residues to enzyme function and will inform subsequent studies of structure-function relationships.

I The properties of the aldosterone antagonists spironolactone and potassium canrenoate in tablet formulations were examined in two studies in healthy subjects, the first study comparing levels of their common major metabolite, canrenone, in plasma and the second study comparing the pharmacological activity of the two drugs in reversing the renal effects of the synthetic mineralocorticoid fludrocortisone. 2 At equal dosage by weight potassium canrenoate yielded peak levels and areas under the curve for canrenone in plasma which were significantly lower than those for spironolactone, and the peak level of canrenone was reached significantly later. Comparison with published work suggests that the experimental formulation of potassium canrenoate had low bioavailability. 3 Spironolactone produced statistically valid log dose-response curves against fludrocortisone as regards sodium excretion, potassium retention and increased urine sodium to potassium ratio. There was no significant log dose-reponse after potassium canrenoate, and a statistically valid estimate of relative potency could not be obtained. 4 The results of the two studies seem inconsistent with the view that canrenone alone is responsible for the pharmacological activity of both drugs, and suggest that a significant part of the activity of spironolactone may be attributable to metabolites other than canrenone.

The study confirms the genetic variability of the CYP11B2 gene and provides us with additional valuable structure-function information.