Modulation of Immunity and Inflammation by the Mineralocorticoid Receptor and Aldosterone.

The mineralocorticoid receptor (MR) is a ligand dependent transcription factor. MR has been traditionally associated with the control of water and electrolyte homeostasis in order to keep blood pressure through aldosterone activation. However, there is growing evidence indicating that MR expression is not restricted to vascular and renal tissues, as it can be also expressed by cells of the immune system, where it responds to stimulation or antagonism, controlling immune cell function. On the other hand, aldosterone also has been associated with proinflammatory immune effects, such as the release of proinflammatory cytokines, generating oxidative stress and inducing fibrosis. The inflammatory participation of MR and aldosterone in the cardiovascular disease suggests an association with alterations in the immune system. Hypertensive patients show higher levels of proinflammatory mediators that can be modulated by MR antagonism. Although these proinflammatory properties have been observed in other autoimmune and chronic inflammatory diseases, the cellular and molecular mechanisms that mediate these effects remain unknown. Here we review and discuss the scientific work aimed at determining the immunological role of MR and aldosterone in humans, as well as animal models.

High sodium intake is associated with increased glucocorticoid production, insulin resistance and metabolic syndrome.

OBJECTIVE: High sodium (HS) diet is associated with hypertension (HT) and insulin resistance (IR). We evaluated whether HS diet was associated with a dysregulation of cortisol production and metabolic syndrome (MetS). PATIENTS AND MEASUREMENTS: We recruited 370 adults (18-85 years, BMI 29·3 ± 4·4 kg/m(2) , 70% women, 72% HT, 61% MetS). HS diet (urinary sodium >150 mEq/day) was observed in 70% of subjects. We measured plasma hormones, lipid profile, urinary free cortisol (UFC) and cortisol tetrahydrometabolites (THM). RESULTS: Urinary sodium was correlated with UFC (r = +0·45, P < 0·001), cortisol THM (r = +0·41, P < 0·001) and inversely with adiponectin, HDL and aldosterone, after adjusting by age, gender and BMI. Subjects with high, compared with adequate sodium intake (50-149 mEq/day) had higher UFC (P < 0·001), THM (P < 0·001), HOMA-IR (P = 0·04), HT (81% vs 50%, P < 0·001), MetS (69% vs 41%, P < 0·001) and lower adiponectin (P = 0·003). A multivariate predictive model adjusted by confounders showed a high discriminative capacity for MetS (ROC curve 0·878) using four clinical variables: HS intake [OR = 5·6 (CI 2·3-15·3)], HOMA-IR [OR 1·7 (1·3-2·2)] cortisol THM [OR 1·2 (1·1-1·4)] and adiponectin [OR = 0·9 (0·8-0·9)], the latter had a protective effect. CONCLUSIONS: High sodium diet was associated with increased urinary cortisol and its metabolites. Also, HS diet was associated with HT, insulin resistance, dyslipidaemia and hypoadiponectinaemia, even when adjusting by confounding variables. Further, we observed that high salt intake, IR and higher cortisol metabolites, alone or combined in a clinical simple model, accurately predicted MetS status, suggesting an additive mechanism in obesity-related metabolic disorders.

Immune system alterations by aldosterone during hypertension: from clinical observations to genomic and non-genomic mechanisms leading to vascular damage.

Hypertension is traditionally considered a disease in which elevated blood pressure contributes to inflammation and activation of the immune system, leading to cardiovascular injury and end-organ damage. Here, we discuss the effects of aldosterone on the immune system and aldosterone's contribution to vascular pathogenesis. Studies in human have suggested a broader role for aldosterone, beyond elevating blood pressure. Recent clinical data support the notion that aldosterone can directly alter the function of the immune system and cause vascular-damaging inflammation. Clinical observations have been reproduced in experimental models of hypertension, further supporting the idea that an aberrant immune response contributes to the onset of hypertension. Such studies have shown that myeloid cells are required to induce the disease and IL-17-producing CD4(+) T cells may contribute to maintaining aldosterone-mediated hypertension. In addition, regulatory T cells diminish the inflammatory damage caused by aldosterone during hypertension. This is a very active area of research that could lead to new therapeutic targets for treating hypertension.

A de novo unequal cross-over mutation between CYP11B1 and CYP11B2 genes causes familial hyperaldosteronism type I.

UNLABELLED: Familial hyperaldosteronism type I (FH-I) is an autosomal dominant disorder caused by an unequal cross-over of the gene encoding steroid 11ß-hydroxylase (CYP11B1) and aldosterone synthase (CYP11B2), giving rise to a chimeric CYP11B1/CYP11B2 gene that displays aldosterone synthase activity regulated by ACTH instead of angiotensin II. AIM: To report an unprecedented case of a de novo unequal crossover mutation between CYP11B1 and CYP11B2 genes causing FH-I. PATIENTS AND METHODS: The index case is a 45-yr-old Chilean male diagnosed with primary aldosteronism (PA). All family members were also studied: his biological parents, 1 brother, 6 sisters, 2 daughters, and 1 son. Plasma renin activity, serum aldosterone, and its ratio were measured in all patients. Genetic analyses were performed using long-extension PCR (XL-PCR), DNA sequencing and Southern blot methods. RESULTS: PA was diagnosed for the index case, 1 of his daughters, his son but not for his parents or siblings. XLPCR and Southern blotting demonstrated the presence of the chimeric CYP11B1/CYP11B2 gene solely in PA-affected subjects, suggesting a case of a de novo mutation. Sequence analysis showed the unequal cross-over CYP11B1/CYP11B2 at intron 2 (c.2600-273 CYP11B2). We also identified a polymorphism at the same intron (c.2600-145C>A CYP11B2) in the genome of the index case's father. CONCLUSION: We describe an unprecedented case of unequal cross-over mutation for the chimeric CYP11B1/CYP11B2 gene causing FH-I, which may be linked to a polymorphism in the index case's father germ line.

Primary aldosteronism can alter peripheral levels of transforming growth factor beta and tumor necrosis factor alpha.

UNLABELLED: Primary aldosteronism (PA) is the most common secondary cause of hypertension that has recently been implicated in alterations of the immune system and progression of cardiovascular disease. OBJECTIVE: To study the cytokines transforming growth factor beta1 (TGF-beta1), tumor necrosis factor alpha (TNF-alpha), and interleukin 10 (IL-10) in patients with PA and essential hypertensives (EH) and evaluate its association with the renin-angiotensin-aldosterone system. PATIENTS AND METHODS: We studied 26 PA and 52 EH patients as controls, adjusted by their blood pressure, body mass index, age, and gender. In both groups, PA and EH, we measured serum aldosterone (SA), plasma renin activity (PRA), and cytokines TGF- beta1, TNF-alpha, and IL-10. In addition, 17 PA patients were treated for 6 months with spironolactone, a mineralocorticoid receptor (MR) antagonist. RESULTS: PA patients had lower levels of TGF-beta1 (17.6+/-4.1 vs 34.5+/-20.5 pg/ml, p<0.001) and TNF-alpha (17.0+/-4.4 vs 35.6+/-21.7 pg/ml, p<0.001) and similar IL-10 levels (99.7+/-18.7 vs 89.4+/-49.5 pg/ml, p: ns), as compared with EH controls. TGF-beta1 and TNF-alpha levels showed a remarkable correlation with SA/PRA ratio in the total group (PA+EH). The treatment of PA patients with spironolactone increased the TGF-beta1 levels (18.3+/-5.9 to 28.4+/-6.3 pg/ml, p<0.001), while TNF-alpha, and IL-10 remained unchanged. CONCLUSION: Our results showed that PA patients have lower TGF-beta1 and TNF-alpha cytokine serum levels than EH. TGF-beta1 levels were restored with spironolactone, showing a MR-dependent regulation. In this way, the chronic aldosterone excess modifies the TGF-beta1 levels, which could produce an imbalance in the immune system homeostasis that may promote an early proinflammatory cardiovascular phenotype.

New splicing mutation of MEN1 gene affecting the translocation of menin to the nucleus.

UNLABELLED: Multiple endocrine neoplasia type 1 (MEN1) is a syndrome inherited in an autosomal dominant trait caused by the inactivation of the tumor suppressor gene MEN1. OBJECTIVE: To communicate a family with a new heterozygous germ line mutation in the intronic region of MEN1 gene and to study its influence in the menin expression. PATIENTS AND METHODS: We studied 5 members of a family with symptomatic hyperparathyroidism (HPT). One of them had also a neuroendocrine pancreatic tumor, and 2 had non-functional multinodular cortical adrenal hyperplasia compatible with the diagnosis of MEN1. After the mutation was identified, HSP92II restriction enzyme was used to determine both zygosity and segregation of the mutation. RT-PCR from leukocyte's isolated mRNA and western blot from pancreatic tumor tissue were performed. In vitro studies were done in Chinese hamster ovary (CHO) cells transfected with reporter minigenes carrying the coding regions spanning exon (EX)-intron 9 and EX10 with the mutant and the wild type sequences. RESULTS: We identified a heterozygous G-to-T substitution in the intron-EX junction (IVS9-1 G>T) of MEN1 gene in the index case and the family members. The mRNA from patient's leukocytes was larger (934 bp) in comparison to the normal transcript (717 bp). Immunoblot analysis demonstrated that wild type (67 kDa) and two additional mutant proteins (approximately 55 and approximately 90 kDa) were expressed in the pancreatic tissue. The in vitro study showed a 45% nuclear localization of the mutated menin signal and a 95% in the wild type protein. CONCLUSIONS: We identified a new intronic heterozygous germ line mutation (IVS9-1G>T) of MEN1 gene in a family affected by MEN1 syndrome. This mutation alters the splice acceptor site of intron 9 that promotes an incorrect splicing, generating aberrant proteins without the nuclear localization signals necessary for the normal menin translocation to the nucleus.

Genetic study of patients with dexamethasone-suppressible aldosteronism without the chimeric CYP11B1/CYP11B2 gene.

Glucocorticoid-remediable aldosteronism is an inherited disorder caused by a chimeric gene duplication between the CYP11B1 (11beta-hydroxylase) and CYP11B2 (aldosterone synthase) genes. The disorder is characterized by hyperaldosteronism and high levels of 18-hydroxycortisol and 18-oxocortisol, which are under ACTH control. The diagnosis of glucocorticoid-remediable aldosteronism had been traditionally made using the dexamethasone suppression test; however, recent studies have shown that several patients with primary aldosteronism and a positive dexamethasone suppression test do not have the chimeric CYP11B1/CYP11B2 gene. The aim of this work was to evaluate whether other genetic alterations exist in CYP11B genes (gene conversion in the coding region of CYP11B1 or in the promoter of CYP11B2) that could explain a positive dexamethasone suppression test and to determine another genetic cause of glucocorticoid-remediable aldosteronism. We also evaluated the role of 18-hydroxycortisol as a specific biochemical marker of glucocorticoid-remediable aldosteronism. We studied eight patients with idiopathic hyperaldosteronism, a positive dexamethasone suppression test, and a negative genetic test for the chimeric gene. In all patients we amplified the CYP11B1 gene by PCR and sequenced exons 3-9 of CYP11B1 and a specific region (-138 to -284) of CYP11B2 promoter. We also measured the levels of 18-hydroxycortisol, and we compared the results with those found in four subjects with the chimeric gene. None of eight cases showed abnormalities in exons 3-9 of CYP11B1, disproving a gene conversion phenomenon. In all patients a fragment of 393 bp corresponding to a specific region of the promoter of CYP11B2 gene was amplified. The sequence of the fragment did not differ from that of the wild-type promoter of the CYP11B2 gene. The 18-hydroxycortisol levels in the eight idiopathic hyperaldosteronism patients and four controls with chimeric gene were 3.9 +/- 2.3 and 21.9 +/- 3.5 nmol/liter, respectively (P < 0.01). In summary, we did not find other genetic alterations or high levels of 18-hydroxycortisol that could explain a positive dexamethasone suppression test in idiopathic hyperaldosteronism. We suggest that the dexamethasone suppression test could lead to an incorrect diagnosis of glucocorticoid-remediable aldosteronism.

Mutations in the CYP21 B gene in a Chilean population with simple virilizing congenital adrenal hyperplasia.

Steroid 21-hydroxylase deficiency (21OHD) compromises about 95% of all cases of congenital adrenal hyperplasia. We have characterized the disease-causing mutations in the steroid 21-hydroxylase genes of 19 Chilean patients (12 females and 7 males) with the simple virilizing (SV) form of 21OHD and compared them with other SV-populations. Using allele-specific polymerase chain reaction, we identified lesions in 28 chromosomes out of 38 tested (73.7%). The most frequent finding was the mutation I173N=12/38 (31.6%) similar as described in Caucasian, Asian and other Hispanic populations, where this mutation represents around 20-40% of the genetic defects in the CYP21B gene. The mutation V282L=4/38 (10.5%) and deletion (Del) or large gene conversion (LGC)=3/38 (7.9%) were also frequently detected. Only 2 alleles carried the mutation I2 splice (5.3%), this frequency is lower than that reported in Caucasian or in Mexican populations. We did not find alleles with the mutations R357W, Cluster E6, P31L and P454S in these patients. The complete genotype was determined in 11/19 patients (58%) and one allele in 6/19 patients (31.6%). In summary, about 30% of the Chilean population with SV 21OHD presented the missense mutation I173N as described in other populations. The frequency of the other lesions showed differences even between populations with similar genetic background.

Primary hyperaldosteronism in essential hypertensives: prevalence, biochemical profile, and molecular biology.

There is evidence that primary aldosteronism (PA) may be common in patients with essential hypertension (EH) when determinations of serum aldosterone (SA), plasma renin activity (PRA), and the SA/PRA ratio are used as screening. An inherited form of primary hyperaldosteronism is the glucocorticoid-remediable aldosteronism (GRA) caused by an unequal crossing over between the CYP11B1 and CYP11B2 genes that results in a chimeric gene, which has aldosterone synthase activity regulated by ACTH. The aim of this study was to evaluate the prevalence of PA and the GRA in 305 EH patients and 205 normotensive controls. We measured SA (1-16 ng/dL) and PRA (1-2.5 ng/mL x h) and calculated the SA/PRA ratio in all patients. A SA/PRA ratio level greater than 25 was defined as being elevated. PA was diagnosed in the presence of high SA levels (>16 ng/dL), low PRA levels (<0.5 ng/mL x h), and very high SA/PRA ratio (>50). Probable PA was diagnosed when the SA/PRA ratio was more than 25 but the other criteria were not present. A Fludrocortisone test was done to confirm the diagnosis. GRA was differentiated from other forms of PA by: the aldosterone suppression test with dexamethasone, the high levels of 18-hydroxycortisol, and the genetic detection of the chimeric gene. In EH patients, 29 of 305 (9.5%) had PA, 13 of 29 met all the criteria for PA, and 16 of 29 were initially diagnosed as having a probable PA and confirmed by the fludrocortisone test. Plasma potassium was normal in all patients. The dexamethasone suppression test was positive for GRA in 10 of 29 and 18-hydroxycortisol levels were high in 2 of 29 patients who had also a chimeric gene. In normotensive subjects, 3 of 205 (1.46%) had PA, and 1 of 205 had a GRA. In summary, we found a high frequency of normokalemic PA in EH patients. A high proportion of PA suppressed SA with dexamethasone, but only a few had a chimeric gene or high levels of 18-hydroxycortisol. These results emphasize the need to further investigate EH patients.

[Hypertension and mineralocorticoids. Usefulness of renin and aldosterone measurements]. / Hipertensión arterial y mineralocorticoides: utilidad de las mediciones de renina y aldosterona.

Recently, some genetic forms of hypertension have been well characterized. These forms can be globally called mineralocorticoid hypertension and are due to different alterations of the renin-angiotensin-aldosterone system (SRAA). Among these, classic primary hyperaldosteronism and its glucocorticoid remediable variety, in which hypertension is secondary to aldosterone production, must be considered. There are also conditions in which mineralocorticoid activity does not depend on aldosterone production. These conditions generate a hyporeninemic hyperaldosteronism, observed in Liddle syndrome, apparent mineralocorticoid hypertension, 11- and 17-hydroxylase deficiency, among others. The detection of these forms of hypertension is only feasible if the renin-angiotensin-aldosterone system is assessed, measuring renin and aldosterone levels. This article reviews these forms of hypertension, their clinical workup and their relevance in the usual hypertensive patients.

Salt-wasting congenital adrenal hyperplasia: detection of mutations in CYP21B gene in a Chilean population.

The steroid 21-hydroxylase deficiency (21OHD) is the most frequent cause of congenital adrenal hyperplasia. We have characterized the disease-causing mutations in the 21-hydroxylase genes of 63 patients with salt-wasting congenital adrenal hyperplasia from a Chilean population of Hispanic origin, a group that has been scarcely evaluated. Using allele-specific PCR, lesions were identified in 97 chromosomes out of 126 tested (77%). The most frequent findings were the gene deletion or large gene conversion (LGC) = 22.9%, I2 splice = 19%, R357W = 12.7%, and Q319X = 10.5%. We did not find alleles with the mutation F308insT and we found three alleles with the cluster E6. The frequency of the point mutation R357W was at least two times more frequent than the one found in Caucasians populations, but similar to that communicated in Asian populations; this finding may be explained by the Asian ancestry of our South-Amerindian population. The frequency of Q319X was also high, similar only to those patients studied in Italy and in a neighboring Argentinian population. In summary, this is a genetic characterization of 21OHD made in an almost pure Hispanic population in Latin America. The high frequency of deletion of CYP21B gene, I2 splice, R357W, and Q319X mutations probably reflects the European-Caucasian-Spanish influence of the conquerors, mixed with Amerindians of Asian ancestry and modulated by other European immigrations.

T235 variant of the angiotensinogen gene and blood pressure in the Chilean population.

BACKGROUND: The angiotensinogen gene has recently been linked to essential hypertension. A variant within this gene, encoding threonine rather than methionine at amino acid position 235, was associated with essential hypertension. However, results of new studies have not confirmed this association, suggesting that ethnic differences may explain the different results. OBJECTIVE: To evaluate whether the T235 variant is associated with a higher incidence of essential hypertension among Hispanics (a group that has scarcely been evaluated) and to determine whether T235 is associated with variations in the plasma renin activity or the serum aldosterone level. PATIENTS AND METHOD: We studied 64 patients with essential hypertension and 62 normotensives, matched for age and sex. We obtained samples for determinations of plasma renin activity, serum aldosterone level and genome DNA from all subjects. The genomic DNA was amplified using the polymerase chain reaction technique and digested by the restriction enzyme streptococcus faecalis (Sfa NI) which cuts M235 only, not T235. RESULTS: The patients with essential hypertension had a higher prevalence of the risk variant T235 (alleles 77/128 = 60.2%) than did the normotensive controls (alleles 65/124 = 52.4%), but the difference was not statistically significant (chi2=1.53, P=0.22). The plasma renin activity levels in hypertensives were not statistically different for homozygous T235, heterozygous and homozygous M235 (1.0 +/- 0.96, 2.0 +/- 2.25 and 1.55 +/- 1.49 ng/ml per h, respectively, P=0.5 1). However, when we considered those hypertensives with low plasma renin activity levels (< 1 ng/ml per h), we found a high prevalence (72.7%) of subjects homozygous for the T235 variant. We found no association between the T235 variant and the serum aldosterone levels in hypertensive and normotensive subjects. CONCLUSIONS: We demonstrated that there is a high prevalence of T235 variant in our Hispanic population. The slight difference between prevalences of T235 variant among hypertensive and normotensive subjects that we found was not statistically significant and did not permit us to establish an association between T235 variant and essential hypertension. We believe that only studying a larger cohort of subjects could show whether there is a quantitative effect of the T allele on plasma renin activity levels.

Genetic variation in P450c11AS in Chilean patients with low renin hypertension.

Low renin hypertension (LRH), which accounts for 10-20% of patients with idiopathic "essential" hypertension, bears hormonal similarities to mineralocorticoid-induced hypertension, but elevated mineralocorticoid concentrations have not been found. Some patients with LRH have normal, rather than suppressed, plasma aldosterone concentrations, so that the ratio of aldosterone concentration to PRA (Aldo/PRA) is high, suggesting inappropriately increased aldosterone biosynthesis. We characterized the CYP11B2 gene that encodes the aldosterone synthase, P450c11AS, in hypertensive and control populations in a single clinic in Santiago, Chile. We directly sequenced the entire CYP11B2 gene in 12 patients with LRH, 2 high renin hypertensive controls, and 2 normotensive controls. All sequences were identical, except that 8 of 24 LRH alleles encoded arginine rather than lysine at position 173. The Arg173 and Lys173 variants were expressed in transfected MA-10 cells, and their ability to convert deoxycorticosterone to aldosterone was measured; the apparent Michaelis constant (Km) for Lys173 was 2.73 mumol/L; the Km for Arg173 was 2.53 mumol/L. The apparent maximal velocity (Vmax) for Lys173 was 6.5 x 10(-3) micrograms/mL.24 h; the Vmax for Arg173 was 7.8 x 10(-3) micrograms/mL.24 h. The first order rate constant, Vmax/Km was 2.38 for Lys173 and 3.08 for Arg173. As these values were not significantly different, we sought to determine whether Arg173 is a polymorphism linked to LRH. We examined position 173 in 52 unselected patients with idiopathic hypertension and 55 normotensive controls by PCR amplification of CYP11B2 exons 3-5 followed by digestion with Bsu361, which digests the Arg173 sequence, but not the Lys173 sequence. More of the hypertensive alleles (39 of 104, 37.5%) than normotensive alleles (25 of 110, 22.5%) carried Arg173 (chi 2 = 5.57; P < 0.02). Most of the Arg173 alleles (31 of 72, 43.1%) were from hypertensive patients with Aldo/PRA below 30, whereas only 5 of 24 (20.8%) Arg173 alleles were found in patients with Aldo/PRA greater than 30 (chi 2 = 3.79; P = 0.05) Thus, the ARg173 variant of CYP11B2 may be linked to LRH in Chilean patients.

Gene conversion in the CYP11B2 gene encoding P450c11AS is associated with, but does not cause, the syndrome of corticosterone methyloxidase II deficiency.

Cytochrome P450c11AS (aldosterone synthase) has 11 beta-hydroxylase, 18-hydroxylase, and 18-oxidase activities and is expressed solely in the adrenal zona glomerulosa. Corticosterone methyloxidase II (CMOII) deficiency denotes a rare disorder of adrenal steroidogenesis in which only the 18-oxidase activity of P450c11AS is disrupted, while the 11 beta-hydroxylase and 18-hydroxylase activities persist. Such patients have elevated serum concentrations of corticosterone and 18-hydroxycorticosterone and very low or unmeasurable concentrations of aldosterone, often resulting in a clinical salt-losing crisis in infancy. One pair of point mutations, Arg181-->Trp and Val386-->Ala, has been previously characterized to cause this disorder in an inbred Iranian Jewish population. We have sought mutations causing CMOII deficiency in outbred populations. In three of four unrelated P450c11AS alleles from two unrelated patients with CMOII deficiency, we found a gene conversion event in which exons 3 and 4 of the CYP11B2 gene encoding P450c11AS were changed to the sequence of the nearby CYP11B1 gene, which encodes the related enzyme P450c11 beta. This conversion resulted in a mutant P450c11AS protein carrying three changes: Asp141-->Glu, Lys151-->Asn, and Ile246-->Thr. We built seven vectors expressing P450c11AS carrying each mutation singly, each of the three possible pairs of mutations, and the triple mutation as found in the proband. The activities of both the normal P450c11AS and the various mutants in transfected nonsteroidogenic COS-1 cells were very low, but their activities in steroidogenic MA-10 and JEG-3 cells were 10- to 20-fold higher. In these systems all of the mutants retained normal 18-oxidase activity, indicating that the detected gene conversion event is associated with but does not cause CMOII deficiency. None of the four CYP11B2 alleles in these two patients bore other identifiable mutations. These patients might have mutations in the promoters or other noncoding regions, or mutations in genes other than CYP11B2 may cause the syndrome of CMOII deficiency.

Molecular biology of mineralocorticoid metabolism.

Mineralocorticoids are adrenal steroid hormones that regulate the retention of sodium by the kidney and, hence, are crucial in the regulation of sodium balance, intravascular volume, and blood pressure. The molecular biology of mineralocorticoid biosynthesis and action has only recently been elucidated. The genes encoding the various enzymes that convert cholesterol to mineralocorticoids have now been cloned. This has revealed the molecular basis of several inherited forms of mineralocorticoid excess, which cause hypertension, and several forms of mineralocorticoid deficiency, which cause salt loss. The cloning of the mineralocorticoid receptor revealed a paradox. Both the mineralocorticoid and the glucocorticoid receptor are activated equally by cortisol, even though cortisol has very modest mineralocorticoid activity. This is explained by the cloning of two genes for the enzyme 11 beta-hydroxysteroid dehydrogenase (11 beta HSD). Type-II 11 beta HSD, found primarily in the kidney, irreversibly converts cortisol to cortisone, which does not activate the mineralocorticoid receptor. Type-II 11 beta HSD thus defends the mineralocorticoid receptor from being activated by the very high concentrations of cortisol in the blood. Recent studies in genetically hypertensive rats suggest that other enzymes or factors that regulate salt balance may remain undiscovered. Thus the study of mineralocorticoid biosynthesis and action remains one of the most promising approaches to understanding hypertension.

Mutation T318M in the CYP11B2 gene encoding P450c11AS (aldosterone synthase) causes corticosterone methyl oxidase II deficiency.

Corticosterone methyl oxidase (CMO) deficiency refers to disorders of aldosterone synthesis due to mutations in the CYP11B2 gene encoding cytochrome P450c11AS, which is the adrenal aldosterone synthase. Type I CMO deficiency is associated with low concentrations of 18OH-corticosterone and aldosterone, due to severe mutations in P450c11AS; while type II CMO deficiency is associated with high concentrations of 18OH-corticosterone and low concentrations of aldosterone, due to less severe mutations of P450c11AS. A single type of mutation, compound homozygosity for R181W and V386A, has been reported as the cause of CMOII deficiency in an inbred population. We now report a patient with a typical clinical and hormonal picture of CMOII deficiency. Direct sequencing of patient and parent DNAs showed that the mother's allele contributed R181W and the deletion/frameshift mutation delta C372, while the father's allele contributed T318M and V386A. These mutants were recreated in cDNA expression vectors singly and in the parental pairs, showing that neither allele contributed any measurable activity. This would suggest the patient should have CMOI deficiency. These studies suggest that other factors besides P450c11AS are involved in the genesis of the distinctive CMOI and CMOII phenotypes.

Artificial mutations in P450c11AS (aldosterone synthase) can increase enzymatic activity: a model for low-renin hypertension?

The conversion of 11-deoxycorticosterone (DOC) to aldosterone is catalyzed by a single enzyme, termed P450c11AS, which has 11 beta-hydroxylase, 18-hydroxylase and 18-oxidase activities. The normotensive Dahl salt-resistant (R) rat has two mutation in P450c11AS that increase its aldosterone synthase activity. If such a mutation were to occur in human patients the predicted phenotype would be low-renin hypertension with elevated ratios of plasma aldosterone to plasma renin activity. Before searching for P450c11AS mutations in such patients we sought to determine if mutations in human P450c11AS could increase enzymatic activity in a fashion analogous to the Dahl R rat. We used site-directed mutagenesis of the human P450c11AS cDNA to create the mutants Glu 136-->Asp, Lys 251-->Arg and the combination of the two; these mutations correspond to those seen in the Dahl R rat. Cells transfected with these mutant human P450c11AS sequences could convert [14C]DOC to corticosterone, 18OH-corticosterone, and aldosterone. In particular the Lys 251-->Arg mutant produced 4 times as much 18OH-corticosterone and 50-80% more aldosterone than the wild type. These data show that mutations of human P450c11AS can increase enzymatic activity, suggesting that such mutations could, in theory, be the basis of some forms of human low-renin hypertension.

Point mutation of Arg440 to His in cytochrome P450c17 causes severe 17 alpha-hydroxylase deficiency.

Genetic disorders in the gene encoding P450c17 cause 17 alpha-hydroxylase deficiency. The consequent defects in the synthesis of cortisol and sex steroids cause sexual infantilism and a female phenotype in both genetic sexes as well as mineralocorticoid excess and hypertension. A 15-yr-old patient from Germany was seen for absent pubertal development and mild hypertension with hypokalemia, high concentrations of 17-deoxysteroids, and hypergonadotropic hypogonadism. Analysis of her P450c17 gene by polymerase chain reaction amplification and direct sequencing showed mutation of codon 440 from CGC (Arg) to CAC (His). Expression of a vector encoding this mutated form of P450c17 in transfected nonsteroidogenic COS-1 cells showed that the mutant P450c17 protein was produced, but it lacked both 17 alpha-hydroxylase and 17,20-lyase activities. To date, 15 different P450c17 mutations have been described in 23 patients with 17 alpha-hydroxylase deficiency, indicating that mutations in this gene are due to random events.

Deletion of amino acids Asp487-Ser488-Phe489 in human cytochrome P450c17 causes severe 17 alpha-hydroxylase deficiency.

17 alpha-Hydroxylase deficiency blocks the biosynthesis of cortisol and sex steroids, resulting in mineralocorticoid excess, hypertension, sexual infantilism, and female phenotype in both genetic sexes. The disease is caused by mutations in the gene encoding cytochrome P450c17, which is the single enzyme that mediates both 17 alpha-hydroxylase and 17,20-lyase activities. We report a 14-yr-old patient from Thailand with a classical clinical presentation of this rare disorder. Analysis of her P450c17 gene by polymerase chain reaction amplification and sequencing showed a nine-base deletion, eliminating codons 487-489 (Asp-Ser-Phe) near the carboxy-terminus of P450c17. This deletion creates a BclI site in the mutant DNA, permitting accurate demonstration that the patient was homozygous for this lesion, whereas one parent and two siblings were heterozygous. By use of site-directed mutagenesis, we created a vector that could express this mutated form of P450c17 when transfected into non-steroidogenic COS-1 cells. Such transfected cells produced immunodetectable P450c17 protein, but had no 17 alpha-hydroxylase or 17,20-lyase activity, whereas cells similarly transfected with a vector expressing normal human P450c17 could 17 alpha-hydroxylate either pregnenolone or progesterone and convert 17 alpha-hydroxypregnenolone to dehydroepiandrosterone, showing the presence of both activities. This is the first report of the molecular genetic basis of 17 alpha-hydroxylase deficiency in a Southeast Asian patient.