Endocrine Abstracts (2005) 9 OC31

Functional effects of mutations in the 11beta-hydroxylase (CYP11B1) gene

M Barr, DM Wilkinson, CD Holloway, S Miller, SM MacKenzie, S Kazi, R Fraser, JMC Connell & E Davies


Division of Cardiovascular & Medical Sciences, Western Infirmary, Glasgow, UK.


Up to 15% of unselected patients with hypertension have evidence of inappropriate aldosterone production, identified by a raised ratio of plasma aldosterone to renin (ARR), suggesting that altered regulation of aldosterone synthesis is a key intermediate phenotype in essential hypertension. We previously described an association between the -344C/T 5'UTR polymorphism in the CYP11B2 (aldosterone synthase) gene and hypertension with raised ARR; the same genetic variation associates with reduced adrenal 11beta-hydroxylase efficiency. This polymorphism is not functional, so is likely to be in linkage dysequilibrium with a nearby causal variant. To identify this variant, we screened the coding regions of CYP11B2 and the neighbouring CYP11B1 (11beta-hydroxylase) gene for mutations in hypertensive subjects stratified by high (>750) and low ARR (<750).

No single mutation was associated with high/low ARR. However, we identified several novel CYP11B1 missense mutations in individual patients. The effects of four mutations (L83S, P135S, F139L and L158P) on enzyme function were assessed. Mutations were recreated by site-directed mutagenesis of a pCMV4/CYP11B1 construct and transfected into JEG3 cells. Enzyme activity was assessed by conversion of 11-deoxycortisol to cortisol and 11-deoxycorticosterone to corticosterone, as measured by HPLC.

L158P and L83S completely abolished enzyme activity, while substrate conversion was significantly reduced in F139L and P135S to approximately 30% of wild-type levels.

Our in vitro study identifies the importance of these affected residues to enzyme function and will inform subsequent studies of structure/function relationships. Several CYP11B2 missense mutations were also found in this cohort of patients and their functional impact is being studied. Patients in whom CYP11B1 mutations were identified have no clear phenotype to suggest significant 11beta-hydroxylase deficiency; all were heterozygotes carrying at least one functional allele; no homozygous mutated individuals were identified. However, these data suggest that variation at this locus is relatively common in the general population.

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