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

Department of Endocrinology and Internal Medicine (C.A.C., A.A.G., L.M.M., J.A.M., C.E.F.) and Faculty of Medicine and Center for Genomics and Bioinformatics (A.G., T.O.P.-A.), Pontificia Universidad Católica de Chile, 114-D Santiago, Chile; Department of Nephrology (E.T.L., M.d.P.H., M.P.R.), Hospital San Juan de Dios, Santiago, Chile; and Division of Endocrinology (D.G.R., C.E.G.-S.), University of Mississippi Medical Center and the G. V. Montgomery Veterans Affairs Medical Center, Jackson, Mississippi 39216

Address all correspondence and requests for reprints to: Carlos E. Fardella, M.D., Department of Endocrinology and Metabolism Faculty of Medicine, Pontificia Universidad Católica de Chile, Lira 85, 5th Floor Santiago, Chile. E-mail: cfardella{at}med.puc.cl.

The human microsomal 11ß-hydroxysteroid dehydrogenase type 2 (11ßHSD2) metabolizes active cortisol into cortisone and protects the mineralocorticoid receptor from glucocorticoid occupancy. In a congenital deficiency of 11ß-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 (GACAAC) and a single nucleotide substitution CT in intron 3. Using site-directed mutagenesis, we generated a mutant 11ßHSD2 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 11ßHSD2 protein were detected by RT-PCR and Western blot, respectively. Wild-type and mutant 11ßHSD2 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 CT in intron 3 (IVS3 + 14 CT) 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.

This work was supported by Chilean Grant FONDECYT 1011035.

Abbreviations: 11ßHSD2, 11ß-Hydroxysteroid dehydrogenase type 2; AME, apparent mineralocorticoid excess; CHO-K1, Chinese hamster ovary K1; F/E, cortisol to cortisone; Km, Michaelis-Menten constant; NAD⁺, nicotinamide adenine dinucleotide (oxidized form); PRA, plasma renin activity; SA, serum aldosterone; THF+aTHF/THE, tetrahydrocortisol plus allotetrahydrocortisol to tetrahydrocortisone; TLC, thin-layer chromatography; TM, transmembrane; Vmax, maximal velocity.

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