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 thinlayer 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.