The aldosterone synthase gene encodes the final step in the production of aldosterone. The aldoststerone synthase gene is polymorphic and variants within the gene and the regulatory region have been associated with hypertension and a phenotype of relatively higher level of aldosterone and its metabolites. However to date; none of the polymorphisms in the regulatory region of CYP11B2 have been shown to alter transcription. Seven novel polymorphisms in the promoter region of CYP11B2 have been identified and the aim of this study was to identify those that may cause a change in binding of transcription factors, leading to altered gene expression and the phenotype of hypertension with an increased aldosterone to renin ratio.
Polymorphisms were screened for putative transcription factor binding sites using a bioinformatics database (Transfac®). Based on this data, a polymorphism at position −1651 (C/T) was selected for initial binding studies. Electromobility shift assays performed using nuclear extracts from human adrenal cell line (H295R). The C (wild-type) and T (mutant) showed different patterns of binding and nuclear extracts were incubated with 5′biotinylated double-stranded DNA probes and streptavidin-agarose beads in order to identify the bound proteins. The proteinDNA complexes were separated on SDS-PAGE gel and following trypsin digestion peptides were analysed by tandem mass spectrometry. Two peptides were identified which bound to the T oligo only; Ape1 which functions as a redox factor, maintaining transcription factors in an active reduced state, and HNRNPK, which interacts with RNA polymerase II transcription machinery, and stimulates transcription. We have confirmed differential binding of adrenal nuclear protein extracts to a polymorphism in the promoter of CYP11B2 and identified candidate proteins. This suggests a mechanism by which polymorphisms in the regulatory region of CYP11B2 may produce a phenotype of relative aldosterone excess and hypertension.