A mutation in the cofactor-binding domain of 11beta-hydroxysteroid dehydrogenase type 2 associated with mineralocorticoid hypertension

Renal 11beta-hydroxysteroid dehydrogenase type 2 (11betaHSD2) is an enzyme responsible for the peripheral inactivation of cortisol to cortisone in mineralocorticoid target tissues. Mutations in the gene encoding 11betaHSD2 cause the syndrome of apparent mineralocorticoid excess (AME), an autosomal recessive form of inherited hypertension, in which cortisol acts as a potent mineralocorticoid. The mutations reported to date have been confined to exons 3-5. Here, we describe two siblings, 1 and 2 yr old, who were diagnosed with hypokalemic hypertension and low plasma aldosterone and renin levels, indicating mineralocorticoid hypertension. Analysis of urinary steroid metabolites showed a markedly impaired metabolism of cortisol, with (tetrahydrocortisol + 5alpha-tetrahydrocortisol)/tetrahydrocortisone ratios of 40-60, and nearly absent urinary free cortisone. Although phenotypically normal, the heterozygous parents showed a disturbed cortisol metabolism. Genetic analysis of the HSD11B2 gene from the AME patients revealed the homozygous deletion of six nucleotides in exon 2 with the resultant loss of amino acids Leu(114) and Glu(115), representing the first alteration found in the cofactor-binding domain. The deletion mutant, expressed in HEK-293 cells, showed an approximately 20-fold lower maximum velocity but increased apparent affinity for cortisol and corticosterone. In contrast, two additionally constructed substitutions, Glu(115) to Gln or Lys, showed increased maximal velocity and apparent affinity for 11beta-hydroxyglucocorticoids. Functional analysis of wild-type and mutant proteins indicated that a disturbed conformation of the cofactor-binding domain, but not the missing negative charge of Glu(115), led to the observed decreased activity of the deletion mutant. Considered together, these findings provide evidence for a role of Glu(115) in determining cofactor-binding specificity of 11betaHSD2 and emphasize the importance of structure-function analysis to elucidate the molecular mechanism of AME.     

In vitro expression studies of a novel mutation delta299 in a patient affected with apparent mineralocorticoid excess

Apparent mineralocorticoid excess syndrome (AME) is an autosomal recessive disorder that results in low renin hypertension and other characteristic clinical features. Typical patients present with severe hypertension, hypokalemia, and undetectable aldosterone. Most patients also have low birth weight, failure to thrive, and nephrocalcinosis. The 11betahydroxysteroid dehydrogenase type 2 (11betaHSD2) defect is documented by demonstrating a failure to convert cortisol to cortisone. Here, we report a patient with typical phenotypic features of AME who does not carry any of the previously described mutations in the HSD11B2 gene. This female patient from a consanguineous Pakistani family presented at age 9 yr. She had a low birth weight compared with her siblings and presented with hypertension (225/120 mm Hg), low plasma renin activity, hypokalemic metabolic alkalosis, suppressed aldosterone, and bilateral nephrocalcinosis. Echocardiogram did not reveal left ventricular hypertrophy, and baseline ophthalmological evaluation did not demonstrate hypertensive retinopathy. However, at age 12 yr, she developed mild to moderate hypertensive retinopathy. Biochemical analysis showed an elevated urinary cortisol to cortisone metabolites ratio (tetrahydrocortisol and 5alpha-tetrahydrocortisol/tetrahydrocortisone) of 28 (normal, 0.66-2.44). She had a cortisol secretion rate of 0.43 mg/d (normal, 5-25 mg/d). Sequence analysis of the HSD11B2 gene revealed a novel homozygous delta299 mutation in exon 5. In vitro expression in Chinese hamster ovary cells revealed that this mutation resulted in no activity.     

Genotype-phenotype correlations of mutations and polymorphisms in HSD11B2, the gene encoding the kidney isozyme of 11beta-hydroxysteroid dehydrogenase

Mutations in the HSD11B2 gene encoding the kidney (11-HSD2) isozyme of 11beta-hydroxysteroid dehydrogenase cause the syndrome of apparent mineralocorticoid excess, a form of salt-sensitive hypertension. Enzymatic activities of mutant enzymes measured in cultured cells are correlated with several parameters of clinical severity including urinary steroid product:precursor ratios, age at diagnosis, birth weight and potassium levels, but not with blood pressure. In normals or in subjects with essential hypertension, sensitivity of blood pressure to salt loading is correlated with activity of renal 11-HSD2, as measured by an increase in the ratio of urinary free cortisol/urinary free cortisone (UFF/UFE), and also correlated with length of a CA repeat polymorphism in the first intron of HSD11B2. A functional explanation for these associations remains to be elucidated.     

Does kidney transplantation normalise cortisol metabolism in apparent mineralocorticoid excess syndrome?

The syndrome of apparent mineralocorticoid syndrome (AME) results from defective 11beta-hydroxysteroid dehydrogenase 2 (11beta-HSD2). This enzyme is co-expressed with the mineralocorticoid receptor (MR) in the kidney and converts cortisol to its inactive metabolite cortisone. Its deficiency allows the unmetabolized cortisol to bind to the MR inducing sodium retention, suppression of PRA and hypertension. Thus, the syndrome is a disorder of the kidney. We present here the first patient affected by AME cured by kidney transplantation. Formerly, she was considered to have a mild form of the syndrome (Type II), but progressively she developed renal failure which required dialysis and subsequent kidney transplantation. To test the ability of the transplanted kidney to normalise the patient's cortisol metabolism, we gave, in two different experiments, 25 and 50 mg/day of cortisone acetate or 15 and 30 mg/day of cortisol after inhibition of the endogenous cortisol by synthetic glucocorticoid (methylprednisolone and dexamethasone). The AME diagnostic urinary steroid ratios tetrahydrocortisol+5alphatetrahydrocortisol/tetrahydrocortisone and cortisol/cortisone were measured by gas chromatography/mass spectrometry. Transplantation resulted in lowering blood pressure and in normalization of serum K and PRA. After administration of a physiological dose of cortisol (15 mg/day), the urinary free cortisol/cortisone ratio was corrected (in contrast to the A-ring reduced metabolites ratio), confirming that the new kidney had functional 11beta-HSD2. This ratio was abnormally high when the supra-physiological dose of cortisol 30 mg/day was given. After cortisone administration, the tetrahydrocortisol+5alphatetrahydrocortisol/tetrahydrocortisone ratio resulted normalised with both physiological and supra-physiological doses, confirming that the hepatic reductase activity is not affected. As expected, the urinary free cortisol/cortisone ratio was normal with physiological, but increased after supra-physiological doses of cortisone. The described case indicates a normalisation of cortisol metabolism after kidney transplantation in AME patient and confirms the supposed pathophysiology of the syndrome. Moreover, it suggests a new therapeutic strategy in particularly vulnerable cohorts of patients inadequately responsive to drug therapy or with kidney failure.     

Molecular basis of human salt sensitivity: the role of the 11beta-hydroxysteroid dehydrogenase type 2.

Salt-sensitive subjects (SS) increase their blood pressure with increasing salt intake. Because steroid hormones modulate renal sodium retention, we hypothesize that the activity of the 11beta-hydroxysteroid dehydrogenase type 2 (11betaHSD2) enzyme is impaired in SS subjects as compared with salt-resistant (SR) subjects. The 11betaHSD2 enzyme inactivates 11-hydroxy steroids in the kidney, thus protecting the nonselective mineralocorticoid receptor from occupation by glucocorticoids. We performed an association study using a recently identified single AluI polymorphism in exon 3 and a polymorphic microsatellite marker of the HSD11B2 gene in 149 normotensive white males (37 SS and 112 SR). The activity of the enzyme 11betaHSD2 was assessed by determining the urinary ratio of cortisol (THF+5alphaTHF) to cortisone (THE) metabolites by gas chromatography in all the 37 SS subjects and in 37 age- and body habitus-matched SR volunteers. Mean (THF+5alphaTHF)/THE ratio was markedly elevated in SS subjects compared with SR subjects (1.51 +/- 0.34 vs. 1.08 +/- 0.26, P < 0.00001), indicating enhanced access of glucocorticoids to the mineralocorticoid receptor in SS subjects. In 58% of SS subjects this ratio was higher than the maximum levels in SR subjects. The salt-induced elevation in arterial pressure increased with increasing (THF+5alphaTHF)/THE ratio (r2 = 0.51, P < 0.0001). A total of 12 alleles of the polymorphic microsatellite marker were detected. Homozygosity for the allele A7 was higher in SS subjects than in SR subjects (41 vs. 28%, P < 0.005), whereas the occurrence of the allele A7 with allele A8 was lower in SS subjects than in SR subjects (8 vs. 15%, P < 0.03). The prevalence of salt sensitivity was 35% in subjects with allele A7/A7, whereas salt sensitivity was present in only 9% of the subjects with allele A7/A8. The (THF+5alphaTHF)/THE ratio was higher in subjects homozygous for the A7 microsatellite allele as compared with the corresponding control subjects. The prevalence of the AluI allele was 8.0% in SR subjects and 5.4% in SS subjects and did not correlate with blood pressure. The decreased activity of the 11betaHSD2 in SS subjects indicates that this enzyme is involved in salt-sensitive blood pressure response in humans. The association of a polymorphic microsatellite marker of the gene with a reduced 11betaHSD2 activity suggests that variants of the HSD 11B2 gene contribute to enhanced blood pressure response to salt in humans.     

Late-onset apparent mineralocorticoid excess caused by novel compound heterozygous mutations in the HSD11B2 gene

Mutations in the gene encoding 11beta-hydroxysteroid dehydrogenase type 2, 11beta-HSD2 (HSD11B2), explain the molecular basis for the syndrome of apparent mineralocorticoid excess (AME), characterized by severe hypertension and hypokalemic alkalosis. Cortisol is the offending mineralocorticoid in AME, as the result of a lack of 11beta-HSD2-mediated cortisol to cortisone inactivation. In this study, we describe mutations in the HSD11B2 gene in 3 additional AME kindreds in which probands presented in adult life, with milder phenotypes including the original seminal case reported by Stewart and Edwards. Genetic analysis of the HSD11B2 gene revealed that all probands were compound heterozygotes, for a total of 7 novel coding and noncoding mutations. Of the 7 mutations detected, 6 were investigated for their effects on gene expression and enzyme activity by the use of mutant cDNA and minigene constructs transfected into HEK 293 cells. Four missense mutations resulted in enzymes with varying degrees of activity, all <10% of wild type. A further 2 mutations generated incorrectly spliced mRNA and predicted severely truncated, inactive enzyme. The mothers of 2 probands heterozygous for missense mutations have presented with a phenotype indistinguishable from "essential" hypertension. These genetic and biochemical data emphasize the heterogeneous nature of AME and the effects that heterozygosity at the HSD11B2 locus can have on blood pressure in later life.     

Apparent mineralocorticoid excess syndrome: an overview

Apparent mineralocorticoid excess (AME) syndrome results from defective 11beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD2). This enzyme is co-expressed with the mineralocorticoid receptor (MR) in the kidney and converts cortisol (F) to its inactive metabolite cortisone (E). Its deficiency allows the unmetabolized cortisol to bind to the MR inducing sodium retention, hypokalemia, suppression of PRA and hypertension. Mutations in the gene encoding 11beta-HSD2 account for the inherited form, but a similar clinical picture to AME occurs following the ingestion of bioflavonoids, licorice and carbenoxolone, which are competitive inhibitors of 11beta-HSD2. Reduced 11beta-HSD2 activity may explain the increased sodium retention in preeclampsia, renal disease and liver cirrhosis. Relative deficiency of 11beta-HSD2 activity can occur in Cushing's syndrome due to saturation of the enzyme and explains the mineralocorticoid excess state that characterizes ectopic ACTH syndrome. Reduced placental 11beta-HSD2 expression might explain the link between reduced birth weight and adult hypertension. Polymorphic variability in the HSD11B2 gene in part determines salt sensitivity, a forerunner for adult hypertension onset. AME represents a spectrum of mineralocorticoid hypertension with severity reflecting the underlying genetic defect in the 11beta-HSD2; although AME is a genetic disorder, several exogenous compounds can bring about the symptoms by inhibiting 11beta-HSD2 enzyme. Substrate excess as seen in Cushing's syndrome and ACTH ectopic production can overwhelm the capacity of 11beta-HSD2 to convert F to E, leading up to an acquired form of AME.     

Apparent mineralocorticoid excess, 11beta hydroxysteroid dehydrogenase and aldosterone action Closing one loop, opening another.

The recent cloning of the human enzyme 11beta hydroxysteroid dehydrogenase type 2 (11betaHSD2), and the demonstration of point mutations or deletions in both familial and apparently sporadic cases of apparent mineralocorticoid excess (AME), underlines the importance of this enzyme in excluding glucocorticoids from mineralocorticoid receptors (MR). Although the sodium retention characteristic of AME can thus be explained by absent or very reduced (< 10%) levels of renal 11 betaHSD2 activity, whether or not the enzymatic defect contributes to the elevated blood pressure by mechanisms other than sodium retention remains to be determined.     

A genetic defect resulting in mild low-renin hypertension

Severe low-renin hypertension has few known causes. Apparent mineralocorticoid excess (AME) is a genetic disorder that results in severe juvenile low-renin hypertension, hyporeninemia, hypoaldosteronemia, hypokalemic alkalosis, low birth weight, failure to thrive, poor growth, and in many cases nephrocalcinosis. In 1995, it was shown that mutations in the gene (HSD11B2) encoding the 11β-hydroxysteroid dehydrogenase type 2 enzyme (11β-HSD2) cause AME. Typical patients with AME have defective 11β-HSD2 activity, as evidenced by an abnormal ratio of cortisol to cortisone metabolites and by an exceedingly diminished ability to convert [11-³H]cortisol to cortisone. Recently, we have studied an unusual patient with mild low-renin hypertension and a homozygous mutation in the HSD11B2 gene. The patient came from an inbred Mennonite family, and though the mutation identified her as a patient with AME, she did not demonstrate the typical features of AME. Biochemical analysis in this patient revealed a moderately elevated cortisol to cortisone metabolite ratio. The conversion of cortisol to cortisone was 58% compared with 0–6% in typical patients with AME whereas the normal conversion is 90–95%. Molecular analysis of the HSD11B2 gene of this patient showed a homozygous C→T transition in the second nucleotide of codon 227, resulting in a substitution of proline with leucine (P227L). The parents and sibs were heterozygous for this mutation. In vitro expression studies showed an increase in the K_m (300 nM) over normal (54 nM). Because ≈40% of patients with essential hypertension demonstrate low renin, we suggest that such patients should undergo genetic analysis of the HSD11B2 gene.     

Mutations in the 11β-Hydroxysteroid Dehydrogenase Type II Enzyme Associated with Hypertension and Possibly Stillbirth

The 11β-hydroxysteroid dehydrogenase type II enzyme (11ßHSD2) converts cortisol into cortisone, thus preventing occupation of the non-selective mineralocorticoid receptor by glucocorticoids in the kidney. Placental 11ßHSD2 is also thought to protect the fetus from the high maternal circulating levels of glucocorticoids. Mutations generating inactive enzymes have been described in the HSD11B2 gene in the congenital syndrome of apparent mineralocorticoid excess (AME) — a low renin form of hypertension. Recently, a mutation has been identified in a family with AME and in which there is a high incidence of stillbirths. In this study we have expressed the R374X mutation and show that the mutant is devoid of enzyme activity in intact mammalian cells expressing a significant level of the truncated protein. While this observation elucidates the cause of AME in this family the degree to which R374X also contributes to the higher incidence of failed pregnancies remains to be determined.     

Cortisol metabolism in hypertension

Corticosteroids are critically involved in blood pressure regulation. Lack of adrenal steroids in Addison's disease causes life-threatening hypotension, whereas glucocorticoid excess in Cushing's syndrome invariably results in high blood pressure. At a pre-receptor level, glucocorticoid action is modulated by 11beta-hydroxysteroid dehydrogenases (11beta-HSDs). 11Beta-HSD1 activates cortisone to cortisol to facilitate glucocorticoid receptor (GR)-mediated action. By contrast, 11beta-HSD2 plays a pivotal role in aldosterone target tissues where it catalyses the opposite reaction (i.e. inactivation of cortisol to cortisone) to prevent activation of the mineralocorticoid receptor (MR) by cortisol. Mutations in the 11beta-HSD2 gene cause a rare form of inherited hypertension, the syndrome of apparent mineralocorticoid excess (AME), in which cortisol activates the MR resulting in severe hypertension and hypokalemia. Ingestion of competitive inhibitors of 11beta-HSD2 such as liquorice and carbenoxolone result in a similar but milder clinical phenotype. Epidemiological data suggests that polymorphic variability in the HSD11B2 gene determines salt sensitivity in the general population, which is a key predisposing factor to adult onset hypertension in some patients. Extrarenal sites of glucocorticoid action and metabolism that might impact on blood pressure include the vasculature and the central nervous system. Intriguingly, increased exposure to glucocorticoids during fetal life promotes high blood pressure in adulthood suggesting an early programming effect. Thus, metabolism and action in many peripheral tissues might contribute to the pathophysiology of human hypertension.     

Biochemical and genetic characterization of 11 beta-hydroxysteroid dehydrogenase type 2 in low-renin essential hypertensives

BACKGROUND: The 11beta-hydroxysteroid dehydrogenase type 2 (11betaHSD2) catalyzes the conversion of cortisol (F) to cortisone (E), avoiding the interaction of cortisol with the mineralocorticoid receptor. If it fails, cortisol will stimulate sodium and water reabsorption, increasing the intravascular volume that suppresses renin and secondarily increase the blood pressure. OBJECTIVE: To look for the possible contribution of a decreased ability of 11betaHSD2 to convert cortisol to its inactive metabolite cortisone in the pathogenesis of low renin hypertension (LREH). PATIENTS AND METHODS: We studied 64 LREH patients (plasma renin activity, PRA < 1 ng/ml per h), eighty normo-renin essential hypertensives (NREH) (PRA: 1-2.5 ng/ml per h) and 74 normotensives. Serum aldosterone (SA), F, E and serum F/E ratio was determined in all patients. A serum F/E ratio was considered high when it was higher than X + 2SD from the normotensive value. Cytosine-adenine (CA)-repeat microsatellite region in intron 1 of HSD11B2 gene was genotyped in all patients and normotensives volunteers. In 13 LREH with high F/E ratio we performed HSD11B2 gene sequencing. RESULTS: LREH had serum F/E ratio higher than NREH and normotensive controls (3.6 (2.9-4.3) versus 2.9 (2.2-4.3) versus 3.0 (2.4-3.7) (P = 0.004), respectively). We observed an inverse relation between F/E ratio and SA and PRA. In NREH and normotensives we did not find correlation between these variables. In the LREH subset the longer 155 bp CA-allele showed the highest serum F/E ratio. No mutations in coding region or short introns were found in LREH patients. CONCLUSION: In this study we show that low-renin essential hypertensives had increased serum cortisol/cortisone ratios as compared with normotensive subjects. This suggest that some essential hypertensives, with suppressed renin activity, may have an impairment in the cortisol inactivation catalyzed by the enzyme 11betaHSD2, whose low activity in LREH patients could be associated with the length of CA-repeat microsatellite in intron 1 of the HSD11B2 gene.     

11beta-hydroxysteroid dehydrogenase and its role in the syndrome of apparent mineralocorticoid excess.

Aldosterone, the most important mineralocorticoid, regulates electrolyte excretion and intravascular volume mainly through its effects on renal cortical collecting ducts, where it acts to increase sodium resorption from and potassium excretion into the urine. Excess secretion of aldosterone or other mineralocorticoids, or abnormal sensitivity to mineralocorticoids, may result in hypokalemia, suppressed plasma renin activity, and hypertension. The syndrome of apparent mineralocorticoid excess (AME) is an inherited form of hypertension in which 11beta-hydroxysteroid dehydrogenase (11-HSD) is defective. This enzyme converts cortisol to its inactive metabolite, cortisone. Because mineralocorticoid receptors themselves have similar affinities for cortisol and aldosterone, it is hypothesized that the deficiency allows these receptors to be occupied by cortisol, which normally circulates at levels far higher than those of aldosterone. We cloned cDNA and genes encoding two isozymes of 11-HSD. The liver or 11-HSD1 isozyme has relatively low affinity for steroids, is expressed at high levels in the liver but poorly in the kidney, and is not defective in AME. The kidney or 11-HSD2 isozyme has high steroid affinity and is expressed at high levels in the kidney and placenta. Mutations in the gene for the latter isozyme have been detected in all kindreds with AME. Moreover, the in vitro enzymatic activity conferred by each mutation is strongly correlated with the ratio of cortisone to cortisol metabolites in the urine, with age of diagnosis, and with birth weight. This suggests that the biochemical and clinical phenotype of AME is largely determined by genotype.     

Molecular basis for the apparent mineralocorticoid excess syndrome in the Oman population

11beta-Hydroxysteroid dehydrogenase type 2 (11beta-HSD2) plays a crucial role in converting hormonally active cortisol to inactive cortisone, thereby conferring specificity upon the mineralocorticoid receptor (MR). Mutations in the gene encoding 11beta-HSD2 (HSD11B2) account for an inherited form of hypertension, the syndrome of "Apparent Mineralocorticoid Excess" (AME) where cortisol induces hypertension and hypokalaemia. We report five different mutations in the HSD11B2 gene in four families from Oman with a total of 9 affected children suffering from AME. Sequence data demonstrate the previously described L114Delta6nt mutation in exon 2 and new mutations in exon 3 (A221V), exon 5 (V322ins9nt) and for the first time in exon 1 (R74G and P75Delta1nt) of the HSD11B2 gene. These additional mutations provide further insight into AME and the function of the 11beta-HSD2 enzyme. The prevalence of monogenic forms of hypertension such as AME remains uncertain. However, our data suggests AME may be a relevant cause of hypertension in certain ethnic groups, such as the Oman population.     

Gas chromatographic-mass spectrometric analysis of urinary glycyrrhetinic acid: an aid in diagnosing liquorice abuse

OBJECTIVES: Liquorice abuse can lead to severe clinical complications, caused by its active compound 18beta-glycyrrhetinic acid (18betaGA). 18betaGA inhibits dehydrogenase activity of 11beta-hydroxysteroid dehydrogenase (11betaHSD). This enzyme catalyses the interconversion between cortisol and cortisone and normally protects the mineralocorticoid receptor from being activated by cortisol. Diagnosing liquorice abuse can be notoriously difficult. The aim of our study was to develop an accurate and clinically applicable 18betaGA urinary assay. DESIGN: We developed a urinary 18betaGA assay based on gas chromatography and mass spectrometry (GCMS) with sufficient sensitivity to detect 18betaGA at low concentrations. The assay was validated in four volunteers consuming different amounts of liquorice. We applied its use in two patients with hypokalaemic hypertension and suppressed plasma renin activity and serum aldosterone, who were suspected of liquorice abuse. RESULTS: The detection limit for 18betaGA of the GC assay was 10 microg L-1, which was lowered to 3 microg L-1 by subsequent application of MS. In all volunteers, urinary 18betaGA was detected during liquorice intake. Urinary 18betaGA remained detectable until 5 days after stopping continued liquorice intake and until at least 51 h after ingestion of a single large amount. Urinary 18betaGA was demonstrated in both patients, establishing a diagnosis of liquorice abuse. One patient showed changes in urinary cortisol metabolites, consistent with 11betaHSD inhibition. Changes in cortisol metabolites were less pronounced in the other patient. CONCLUSION: Liquorice abuse can result in hypokalaemic hypertension with prolonged suppression of plasma renin activity and aldosterone concentration. This is caused by 18betaGA-mediated inhibition of 11betaHSD, resulting in activation of the renal mineralocorticoid receptor by cortisol. Urinary 18betaGA measurement by GCMS is a useful aid in establishing liquorice abuse.     

Two homozygous mutations in the 11 beta-hydroxysteroid dehydrogenase type 2 gene in a case of apparent mineralocorticoid excess

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.     

Apparent mineralocorticoid excess.

Apparent mineralocorticoid excess (AME) is a potentially fatal genetic disorder causing severe juvenile hypertension, pre- and postnatal growth failure, hypokalemia and low to undetectable levels of renin and aldosterone. It is caused by autosomal recessive mutations in the HSD11B2 gene, which result in a deficiency of 11 beta-hydroxysteroid dehydrogenase type 2 (11 beta-HSD2). The 11 beta-HSD2 enzyme is responsible for the conversion of cortisol to the inactive metabolite cortisone and, therefore, protects the mineralocorticoid receptors from cortisol intoxication. In 1998, a mild form of this disease was reported, which might represent an important cause of low-renin hypertension. Early and vigilant treatment might prevent or improve the morbidity and mortality of end-organ damage.     

Hypertension and the cortisol-cortisone shuttle

11 beta-Hydroxysteroid dehydrogenase type 2 (11 beta-HSD2) plays a crucial role in converting hormonally active cortisol to inactive cortisone, thereby conferring specificity on the mineralocorticoid receptor. Mutations in the gene encoding 11 beta-HSD2 (HSD11B2) account for an inherited form of hypertension, the syndrome of apparent mineralocorticoid excess, in which cortisol induces hypertension and hypokalemia. A similar clinical picture to apparent mineralocorticoid excess occurs after the ingestion of licorice and carbenoxolone, which are competitive inhibitors of 11 beta-HSD2. Reduced 11 beta-HSD2 activity may explain the increased sodium retention in preeclampsia, renal disease, and liver cirrhosis. Substrate saturation of 11 beta-HSD2 occurs in Cushing's syndrome and explains the mineralocorticoid excess state that characterizes ectopic ACTH syndrome. Polymorphic variability in the HSD11B2 gene in part determines salt sensitivity, a forerunner for adult onset hypertension. Furthermore, reduced placental 11 beta-HSD2 expression might underpin the Barker hypothesis, the epidemiological link between reduced birth weight and adult hypertension. At a prereceptor level, 11 beta-HSD2 plays a key role in normal physiology in the corticosteroid regulation of sodium homeostasis and pathophysiology of hypertension.     

NEW FINDINGS IN APPARENT MINERALOCORTICOID EXCESS

We report two female siblings (ages 4 and 9 years) and one 8-year-old male with the syndrome of apparent mineralocorticoid excess (AME) presenting with low renin hypertension and hypoaldosteronism. The deficiency of 11β-hydroxysteroid dehydrogenase results in a defect of the peripheral metabolism of Cortisol (F) to cortisone (E). As a result, the serum Cortisol half-life (T½) is prolonged, ACTH is suppressed, and serum F is normal. The specific diagnosis of the disorder was made by the decreased ratio of the urinary metabolites of E (tetrahydrocortisone, THE) and F (tetrahydrocortisol, THF). Continuous i.v. hydrocortisone administration caused an increase in blood pressure and decrease in serum potassium demonstrating the abnormal mineralocorticoid activity of Cortisol in these patients. Addition of spironolactone resulted in a decrease in blood pressure, rise in serum potassium and a gradual increase in plasma renin activity. These studies suggest that an abnormality in Cortisol action or metabolism results in Cortisol behaving as a potent mineralocorticoid and causing the syndrome of AME.