Two Homozygous Mutations in the 11β-Hydroxysteroid Dehydrogenase Type 2 Gene in a Case of Apparent Mineralocorticoid Excess

Abstract

The human microsomal 11β-hydroxysteroid dehydrogenase type 2 (11βHSD2) metabolizes active cortisol into cortisone and protects the mineralocorticoid receptor from glucocorticoid occupancy. In a congenital deficiency of 11β-HSD2, the protective mechanism fails and cortisol gains inappropriate access to mineralocorticoid receptor, resulting in low-renin hypertension and hypokalemia. In the present study, we describe the clinical and molecular genetic characterization of a patient with a new mutation in the HSD11B2 gene. This is a 4-yr-old male with arterial hypertension. The plasma renin activity and serum aldosterone were undetectable in the presence of a high cortisol to cortisone ratio. PCR amplification and sequence analysis of HSD11B2 gene showed the homozygous mutation in exon 4 Asp223Asn (GAC→AAC) and a single nucleotide substitution C→T in intron 3. Using site-directed mutagenesis, we generated a mutant 11βHSD2 cDNA containing the Asp223Asn mutation. Wild-type and mutant cDNA was transfected into Chinese hamster ovary cells and enzymatic activities were measured using radiolabeled cortisol and thin-layer chromatography. The mRNA and 11βHSD2 protein were detected by RT-PCR and Western blot, respectively. Wild-type and mutant 11βHSD2 protein was expressed in Chinese hamster ovary cells, but the mutant enzyme had only 6% of wild-type activity. In silico 3D modeling showed that Asp223Asn changed the enzyme’s surface electrostatic potential affecting the cofactor and substrate enzyme-binding capacity. The single substitution C→T in intron 3 (IVS3 + 14 C→T) have been previously reported that alters the normal splicing of pre-mRNA, given a nonfunctional protein. These findings may determine the full inactivation of this enzyme, explaining the biochemical profile and the early onset of hypertension seen in this patient.