title: Two homozygous mutations in the 11 beta-hydroxysteroid dehydrogenase type 2 gene in a case of apparent mineralocorticoid excess.
abstract: The human microsomal 11 beta-hydroxysteroid dehydrogenase type 2 (11 beta HSD2) metabolizes active cortisol into cortisone and protects the mineralocorticoid receptor from glucocorticoid occupancy. In a congenital deficiency of 11 beta-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 11 beta HSD2 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 11 beta HSD2 protein were detected by RT-PCR and Western blot, respectively. Wild-type and mutant 11 beta HSD2 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.
authors.affiliation: Department of Endocrinology and Internal Medicine, Pontificia Universidad Católica de Chile, 114-D Santiago, Chile.
authors.forename: Cristian A
authors.initials: CA
authors.lastname: Carvajal
authors.forename: Alexis A
authors.initials: AA
authors.lastname: Gonzalez
authors.forename: Damian G
authors.initials: DG
authors.lastname: Romero
authors.forename: Angel
authors.initials: A
authors.lastname: González
authors.forename: Lorena M
authors.initials: LM
authors.lastname: Mosso
authors.forename: Elizabeth T
authors.initials: ET
authors.lastname: Lagos
authors.forename: Maria del P
authors.initials: Mdel P
authors.lastname: Hevia
authors.forename: Maria P
authors.initials: MP
authors.lastname: Rosati
authors.forename: Tomás O
authors.initials: TO
authors.lastname: Perez-Acle
authors.forename: Celso E
authors.initials: CE
authors.lastname: Gomez-Sanchez
authors.forename: Joaquín A
authors.initials: JA
authors.lastname: Montero
authors.forename: Carlos E
authors.initials: CE
authors.lastname: Fardella
mesh_terms: D043209:11-beta-Hydroxysteroid Dehydrogenase Type 2; D000818:Animals; D001216:Asparagine; D001224:Aspartic Acid; D001483:Base Sequence / Q000235:genetics; D016466:CHO Cells; D002675:Child, Preschool; D006224:Cricetinae; D003545:Cysteine; D006720:Homozygote; D006801:Humans; D006913:Hydroxysteroid Dehydrogenases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D008297:Male; D008901:Mineralocorticoids / Q000378:metabolism*; D008958:Models, Molecular; D009154:Mutation; D020641:Polymorphism, Single Nucleotide; D013912:Threonine
chemical_list: D008901:Mineralocorticoids; D013912:Threonine; D001224:Aspartic Acid; D001216:Asparagine; D006913:Hydroxysteroid Dehydrogenases; D043209:11-beta-Hydroxysteroid Dehydrogenase Type 2; C468387:HSD11B2 protein, human; D003545:Cysteine