ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

Endocrine Abstracts (2011) 25 OC2.3

APEX1, a novel, negative regulator of aldosterone synthase activity, differentially binds to a single nucleotide polymorphism in the aldosterone synthase gene and represses transcriptional activity both in vitro and in vivo

Frances McManus1, William Sands1, Robert Fraser1, Eleanor Davies1 & John Connell2

1University of Glasgow, Glasgow, UK; 2University of Dundee, Dundee, UK.

Aldosterone synthesis is heritable; a single nucleotide polymorphism (SNP) in the promoter of the aldosterone synthase gene (position −344, rs1799998) has been associated with an increased plasma aldosterone levels and hypertension. However, deletion of this site has no effect on gene transcription in vitro and therefore the mechanism that links genotype with phenotype is unclear.

We identified a polymorphism at position −1651 T/C (rs13268025) and show it to be in linkage disequilibrium with the −344 SNP. Reporter gene constructs containing contrasting alleles at position −1651 were transfected into H295R cells. The C allele had (75%) greater promoter activity than the T allele. An electromobility shift assay with oligonucleotides spanning the polymorphism of interest demonstrated a difference in protein/DNA complexes between the T and C allele. Biotinylated pull down assays of the protein: DNA complex were proteolised and peptides identified by tandem mass spectroscopy. Fragments derived from the transcription factor APEX1, were identified bound to the T allele and not the C allele. Chromatin immunoprecipitation confirmed the association of APEX1 to the CYP11B2 promoter. Inhibition of APEX1 activity with a small molecule inhibitor of APEX1 (E3330, Sigma–Aldrich), and siRNA for APEX1, both increased transcriptional activity. The in vivo genotype/phenotype relationship was explored in 60 normal volunteers, studied under standard salt intake. Carriers of the C allele were confirmed to have higher 24 h urinary aldosterone excretion (mean=59.55 μg/24 h 95% CI 46.73–72.37 μg/24 h) than the subjects carrying the T allele at position −1651 (mean=36.10 μg/24 h 95% CI 24.0–48.21 μg/24 h) P=0.008.

APEX1 has been identified as a novel negative regulator of CYP11B2; a SNP at −1651 demonstrates allele dependant binding, leading to increased transcriptional activity in vitro and increased THAldo excretion in vivo. This offers a plausible mechanism behind the genotype/phenotype association of increased aldosterone secretion and hypertension.

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