Plasma corticosteroid profiling: brief opinion of its current status in clinical diagnosis and research.

Adrenal steroid biosynthesis comprises a series of dynamically interrelated, enzyme-catalyzed reactions in two separate compartments, the zona glomerulosa and the zona fasciculata/reticularis. End products (cortisol, aldosterone and androgens), together with a proportion of the intermediate compounds, appear in the circulation as a characteristic profile. Rare deficiencies of individual enzymes modify this profile in a recognizable way. Previous exhaustive profiling suggests that their diagnosis can now often be made on the basis of single-compound analyses with concomitant genetic tests. However, high-capacity liquid chromatography coupled with tandem mass spectrometry-based methods are facilitating profiling of large population samples, revealing that smaller differences in enzyme efficiency, indicated by potentially more complex corticosteroid patterns, may be related to clinical wellbeing in a much larger proportion of the population.

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.

Aldosterone synthase gene variation and adrenocortical response to sodium status, angiotensin II and ACTH in normal male subjects.

OBJECTIVE: Aldosterone synthase, a key enzyme in the terminal steps of aldosterone synthesis, is encoded by the CYP11B2 gene. A polymorphism in the 5' coding region of this gene (-344 C/T) is associated with hypertension, particularly with elevation of the aldosterone to renin ratio. A second polymorphism (a conversion in intron 2 to resemble that of the neighbouring 11beta-hydroxylase (CYP11B1) gene) is found in close linkage dysequilibrium with the variant at -344 C/T. The mechanism by which these variants predispose to cardiovascular disease and the precise intermediate phenotype associated with them remains speculative. DESIGN: We performed a focused physiological study in normal volunteers stratified by CYP11B2 genotype. PATIENTS: Twenty-three subjects homozygous for the T allele and 21 homozygous for the C allele of the -344 C/T polymorphism of CYP11B2 were studied. MEASUREMENTS: Basal and angiotensin II stimulated plasma and 24-h urinary steroid excretion during low (60 mmol/day) and high (160 mmol/day) sodium intake and plasma steroids after ACTH stimulation were measured. RESULTS: No influence of polymorphic variation on basal or stimulated plasma cortisol or aldosterone or other plasma steroid concentrations during either dietary phase was seen. However, excretion of tetrahydro-11-deoxycortisol (the urinary metabolite of 11-deoxycortisol), which is the precursor of cortisol) was increased in TT subjects during sodium restriction, consistent with impairment of zona fasciculata 11beta-hydroxylation. CONCLUSIONS: We conclude that this polymorphism has no major influence on normal zona glomerulosa function but is associated with a change in 11beta-hydroxylation in the zona fasciculata. The mechanism remains uncertain, but alteration of 11-deoxycortisol levels without change in cortisol suggests altered efficiency of 11beta-hydroxylation. In the long term, this may lead to a minor but chronic increase in ACTH drive to the gland, which may have consequences for steroid synthesis and predispose to the risk of cardiovascular disease.

The impact of polymorphisms in the gene encoding aldosterone synthase (CYP11B2) on steroid synthesis and blood pressure regulation.

The terminal stages in the synthesis of aldosterone and cortisol are catalysed by the enzymes aldosterone synthase and 11beta-hydroxylase respectively. We have previously reported that polymorphic variation in the 5' promoter region (-344C/T) of the gene encoding aldosterone synthase (CYP11B2) is associated with increased aldosterone metabolite excretion and with hypertension associated with a raised aldosterone to renin ratio (ARR). Additionally, basal and ACTH-stimulated plasma levels of 11-deoxycortisol, the precursor of cortisol, are higher in subjects carrying the T-allelic variant. We have now identified in a family study (573 individuals from 105 extended families ascertained through a hypertensive proband) that excretion of the main metabolite of this steroid (tetrahydro-11-deoxycortisol, THS) is heritable (19.4%) and that the T-allele of CYP11B2 is more strongly associated with higher THS levels than the C-allele. Raised plasma and urinary levels of 11-deoxycortisol suggest that there is relative inefficiency of 11beta-hydroxylation in the zona fasciculata; the P450 enzyme responsible for this step is encoded by the gene CYP11B1, which is highly homologous with and adjacent to CYP11B2. The association of genetic variation in the promoter of CYP11B2 which, in the adrenal cortex, is only expressed in zona glomerulosa, and zona fasciculata 11beta-hydroxylation function is paradoxical. There may be linkage dys-equilibrium between this polymorphism and a quantitative trait locus (QTL) in CYP11B1. Chronic alteration of 11beta-hydroxylase activity may increase ACTH drive to the adrenal cortex, altering the regulation of aldosterone synthesis. This may explain, at least partly, the association between CYP11B2 polymorphisms and hypertension.