Aldosterone synthase (CYP11B2) deficiency among Palestinian infants: Three novel variants and genetic heterogeneity.

Aldosterone synthase deficiency (ASD) is a rare potentially life-threatening genetic disorder that usually presents during infancy due to pathogenic variants in the CYP11B2 gene. Knowledge about CYP11B2 variants in the Arab population is scarce. Here, we present and analyze five Palestinian patients and their different novel pathogenic variants. Data on clinical presentation, electrolytes, plasma renin activity, and steroid hormone levels of five patients diagnosed with ASD were summarized. Sequencing of the CYP11B2 gene exons was followed by evolutionary conservation analysis and structural modeling of the variants. All patients were from highly consanguineous Palestinian families. The patients presented at 1-4 months of age with recurrent vomiting, poor weight gain, hyponatremia, hyperkalemia, and low aldosterone levels. Genetic analysis of the CYP11B2 gene revealed three homozygous pathogenic variants: p.Ser344Profs*9, p.G452W in two patients from an extended family, and p.Q338stop. A previously described pathogenic variant was found in one patient: p.G288S. We described four different CYP11B2 gene pathogenic variants in a relatively small population. Our findings may contribute to the future early diagnosis and therapy for patients with ASD among Arab patients who present with failure to thrive and compatible electrolyte disturbances.

Impact of immunohistochemistry on the diagnosis and management of primary aldosteronism: An important tool for improved patient follow-up.

BACKGROUND AND AIMS: Primary aldosteronism is a common cause of secondary hypertension. Primary aldosteronism is caused by an aldosterone-producing adenoma or bilateral hyperplasia that in some cases is asymmetrical with one adrenal dominating aldosterone secretion. Most patients with aldosterone-producing adenoma are biochemically cured by unilateral adrenalectomy, but patients with bilateral hyperplasia have a significant risk of residual or recurrent disease. Here, immunohistochemistry of CYP11B1 and B2 was used to investigate whether these markers could aid in the diagnostic workup of primary aldosteronism patients. MATERIALS AND METHODS: A total of 39 patients with primary aldosteronism who underwent unilateral adrenalectomy for a presumed adenoma during 2013-2016 were included. Immunohistochemistry using monoclonal antibodies identifying the enzymes CYP11B1 and B2 was part of routine histopathological workup in 6 cases; in 33 cases, it was applied retrospectively. The hyperplasia diagnosis was suggested when there was no dominating nodule but immunoreactivity for CYP11B2 was seen in several nodules, which were also seen on routine sections. To distinguish between adenoma and hyperplasia, a ratio between the largest and second largest CYP11B2-positive nodules was calculated. RESULTS: In all, 22 patients had an aldosterone-producing adenoma, while 13 patients were judged to have hyperplasia. In four cases, a final diagnosis could not be established, thus these were judged equivocal. Among the 33 cases investigated retrospectively, the primary histopathological diagnosis was altered from hyperplasia to aldosterone-producing adenoma in 9 cases (27%) after immunohistochemistry, and the immunohistochemically rectified adenoma group displayed improved clinical cure rates compared to the routine H&E-diagnosed cohort. Moreover, the B2 ratio was significantly higher in adenoma than in hyperplasia and equivocal cases. CONCLUSION: Immunohistochemistry detecting CYP11B1 and B2 expression is of great help in establishing a final histopathological diagnosis in patients with primary aldosteronism. This procedure should be part of the histopathological routine in all operated primary aldosteronism patients.

Steroid Profiling and Immunohistochemistry for Subtyping and Outcome Prediction in Primary Aldosteronism-a Review.

PURPOSE OF REVIEW: Steroid profiling and immunohistochemistry are both promising new tools used to improve diagnostic accuracy in the work-up of primary aldosteronism (PA) and to predict treatment outcomes. Herein, we review the recent literature and present an outlook to the future of diagnostics and therapeutic decision-making in patients with PA. RECENT FINDING: PA is the most common endocrine cause of arterial hypertension and unilateral forms of the disease are potentially curable by surgical resection of the overactive adrenal. Recent studies have shown that adrenal steroid profiling by liquid chromatography-tandem mass spectrometry (LC-MS/MS) can be helpful for subtyping unilateral and bilateral forms of PA, classifying patients with a unilateral aldosterone-producing adenoma (APA) according to the presence of driver mutations of aldosterone production in APAs, and potentially predicting the outcomes of surgical treatment for unilateral PA. Following adrenalectomy, immunohistochemistry of aldosterone synthase (CYP11B2) in resected adrenals is a new tool to analyze "functional" histopathology and may be an indicator of biochemical outcomes after surgery. Biochemical and clinical outcomes of therapy in PA vary widely among patients. Peripheral venous steroid profiling at baseline could improve diagnostic accuracy and help in surgical decision-making in cases of a suspected APA; results of "functional" histopathology could help determine which patients are likely to need close post-surgical follow-up for persistent aldosteronism.

Histopathological and genetic characterization of aldosterone-producing adenomas with concurrent subclinical cortisol hypersecretion: a case series.

PURPOSE: Aldosterone-producing adenomas with concurrent subclinical cortisol hypersecretion are reported in an increasing number of patients. Five aldosterone-producing adenomas from patients with primary aldosteronism and subclinical hypercortisolism were examined. THE AIMS OF OUR STUDY WERE: (1) to analyze pathological features and immunohistochemical expression of CYP11B1 (11ß-hydroxylase) and CYP11B2 (aldosterone synthase) in these tumors; (2) to investigate somatic mutations involved in adrenal steroid hypersecretion and/or tumor growth. METHODS: Archival micro-dissected paraffin-embedded slides from tumor specimens were used for histological and molecular studies. Immunohistochemistry was performed using monoclonal anti-CYP11B1 and anti-CYP11B2 antibodies. Cellular composition was determined by examining for known features of zona fasciculata and zona glomerulosa, and immunoreactivity for CYP11B1 and CYP11B2 by McCarty H-score. Spot regions for mutations in KCNJ5, ATP1A1, ATP2B3, CACNA1D, PRKACA, and CTNNB1 gene sequences were evaluated. RESULTS: Four APAs showed a predominant (≥50%) zona fasciculata-like cell pattern: one tumor had CYP11B1 H-score = 150, no detectable CYP11B2 expression, and harbored a PRKACA p.Leu206Arg mutation (that we have reported previously elsewhere), one had no CYP11B1 expression, CYP11B2 H-score = 40, and no mutations; the remaining two adenomas had high CYP11B1 H-score (160 and 240, respectively) and low CYP11B2 H-score (30 and 15, respectively), with the latter harboring a CTNNB1 p.Ser45Phe activating mutation. One of five aldosterone-producing adenomas had a predominant zona glomerulosa-like pattern, CYP11B1 H-score = 15, CYP11B2 H-score = 180, and no mutations. CONCLUSIONS: The majority of aldosterone-producing adenomas with concurrent subclinical cortisol hypersecretion were composed mainly of zona fasciculata-like cells, while CYP11B1 and CYP11B2 immunostaining demonstrated clear heterogeneity. In a subset of cases, different somatic mutations may be involved in hormone excess and tumor formation.

The impacts of the interaction of genetic variation, CYP11ß2 and NEDD4L, with sodium intake on pediatric obesity with gender difference: a 3-year panel study.

Backgrounds/Objectives:This panel study was to predict the incidences of pediatric obesity by the interaction of sodium (Na) intake and nine single-nucleotide polymorphisms (SNPs) of salt-sensitive genes (SSGs), ACE(angiotensin-converting enzyme), ADD1 G460W,AGT M235T,CYP11ß2 (cytochrome P450 family 11-subfamily ß-2, -aldosterone synthase),GNB3 C285T,GRK4(A142V)(G-protein-coupled receptor kinases type 4),GRK4 (A486V),NEDD4L (neural precursor cell expressed developmentally downregulated 4 like; rs2288774) and SLC12A3 (solute carrier family 12 (Na/Cl transporters)-member 3), selected from genome-wide association study. SUBJECTS/METHODS: Non-obese (non-OB) Korean children of 9 years old were recruited from eight elementary schools in Seoul in 2007 and 2009, each. Follow-up subjects (total=798) in 2010 and 2012 were final participants. Participants were classified as OB group for those whose body mass index were over the 85th percentile using the 'Korean National Growth Charts', and others were classified as non-OB. With nine SNPs typing, the genetic interaction with the variation of Na intake for 3 years was evaluated as an obesity risk. RESULTS: The obesity incidence rate for non-OB children at baseline after 3 years was 10.31%. Na intake in non-OB after 3 years was significantly decreased compared with the baseline, whereas Na intake reduction was undetectable in OB. We found gender differences on association between the changes of Na intake and the obesity incidence for 3 years by the SSG variation. Odds ratio for the obesity risk was 5.75 times higher in girls having hetero/mutant types of NEDD4L with higher Na intakes (Q2+Q3+Q4 in quartiles) compared with that in the wild type with the lowest Na intake (Q1). Girls with hetero/mutant of CYP11ß2 tended to increase the obesity incidence as Na intake increased (Q1

Aldosterone Synthase Promoter Polymorphism and Cardiovascular Phenotypes in a Large, Multiethnic Population-Based Study.

BACKGROUND: A single-nucleotide polymorphism in the aldosterone synthase gene (CYP11B2) promoter [-344C/T, rs1799998] has been reported to associate with cardiovascular phenotypes. METHODS: The Dallas Heart Study is a large, multiethnic cohort with a high prevalence of hypertension. We genotyped 3452 Dallas Heart Study participants for -344C/T. Generalized linear models were used to assess whether variation at -344C/T associated with plasma aldosterone concentration (PAC), systolic and diastolic blood pressure (SBP and DBP), plasma glucose (in persons with no diabetes), HOMA IR (Homeostasis Model Assessment as an Index of Insulin Resistance), and left ventricular (LV) mass indexed to height. Systolic blood pressure and DBP were significantly higher in blacks compared with whites (P < 0.001 for SBP and for DBP) and Hispanics (P < 0.001 for SBP and for DBP). Log-transformed body mass index was also significantly higher in blacks compared with whites (P < 0.001), but not Hispanics (P = 0.10). Log-transformed PAC was higher in whites compared with blacks (P < 0.001), but did not differ significantly in whites compared with Hispanics (P = 0.73). In univariate and multivariable analysis, -344C/T was not significantly associated with PAC within any ethnicity. In univariate and multivariable analysis, -344C/T was not associated with SBP or DBP within any ethnicity. After adjustment for multiple testing, univariate and multivariable analyses revealed no association between -344C/T and plasma glucose in patients with no diabetes, HOMA IR, or LV mass indexed to height. CONCLUSIONS: We were unable to reproduce previously reported associations between -344C/T and PAC, blood pressure, plasma glucose, or LV mass. Methodological differences might explain the differences between our findings and those previously reported.

Primary aldosteronism: functional histopathology and long-term follow-up after unilateral adrenalectomy.

OBJECTIVES: To investigate the long-term outcome after unilateral adrenalectomy in patients with primary aldosteronism (PA) and to establish the role of functional pathology for the final diagnosis of aldosterone-producing adenoma (APA) or hyperplasia. DESIGN: A single-centre, retrospective cohort study in a hospital setting. PATIENTS: Consecutive patients with PA, n = 120, who underwent unilateral adrenalectomy between 1985 and 2010. Preoperative and postoperative data were analysed. To indicate the site of aldosterone secretion in the resected adrenal, we added functional methods to routine histopathology, using in situ hybridization and immunohistochemistry to detect the presence of enzymes needed for aldosterone (CYP11B2) and cortisol (CYP11B1, CYP17A1) synthesis. RESULTS: The median follow-up was 5 years and the cure rate of PA 91%. Hypertension was improved in 97% and normalized in 38%. Functional histopathology changed the final diagnosis from APA to hyperplasia in 6 cases (7%). Five of these had no expression of or staining for aldosterone synthase in the adenoma, but only in nodules in the adjacent cortex. All except one APA patient were cured of PA. They had lower preoperative serum potassium and higher 24-h urinary aldosterone than patients with hyperplasia. Among patients with hyperplasia 16 of 26 were cured. CONCLUSIONS: Most patients were cured of PA by unilateral adrenalectomy. Almost all noncured benefitted from the operation as the blood pressure improved. Functional histopathology proved helpful in the distinction between APA and hyperplasia, and we recommend that functional histopathology should be added to routine histopathology to improve the diagnostic evaluation and aid in tailoring the follow-up.

Modulation of aldosterone levels by -344 C/T CYP11B2 polymorphism and spironolactone use in resistant hypertension.

Interindividual variability in plasma aldosterone levels comprises environmental and genetic sources. Increased aldosterone levels have been associated with higher risk of hypertension and target-organ damage related to hypertension. Aldosterone excess and intravascular volume expansion are implicated in pathophysiology of resistant hypertension (RH). We sought to investigate whether -344 C/T polymorphism (rs1799998) in aldosterone synthase gene (CYP11B2) is associated with plasma aldosterone levels in patients with resistant hypertension. Sixty-two patients with resistant hypertension were enrolled in this cross-sectional study. Genotypes were obtained by allelic discrimination assay using real time polymerase chain reaction. Multivariable linear regression was used to identify whether TT genotype was a predictor of aldosterone levels. No differences in clinical and laboratorial parameters were found among genotype groups. We found an additive effect of the T allele on plasma aldosterone concentration in RH. Also, there was higher aldosterone levels in TT homozygous under use of spironolactone compared with C carriers and compared with TT subjects who was not under use of spironolactone. TT genotype and the use of spironolactone were significant predictors of aldosterone levels in RH subjects. Plasma aldosterone concentration is significantly associated with -344 C/T CYP11B2 polymorphism and with the treatment with spironolactone in resistant hypertensive subjects.

Angiotensin converting enzyme gene polymorphism is associated with severity of coronary artery disease in men with high total cholesterol levels.

This study examines whether renin-angiotensin-aldosterone system gene polymorphisms: ACE (encoding for angiotensin converting enzyme) c.2306-117_404 I/D, AGTR1 (encoding for angiotensin II type-1 receptor) c.1080*86A>C and CYP11B2 (encoding for aldosterone synthase) c.-344C>T are associated with the extension of coronary atherosclerosis in a group of 647 patients who underwent elective coronary angiography. The extension of CAD was evaluated using the Gensini score. The polymorphisms were determined by PCR and RFLP assays. The associations between genotypes and the extent of coronary atherosclerosis were tested by the Kruskal-Wallis test, followed by pairwise comparisons using Wilcoxon test. The population has been divided into groups defined by: sex, smoking habit, past myocardial infarction, BMI (>, ≤ 25), age (>, ≤ 55), diabetes mellitus, level of total cholesterol (>, ≤ 200 mg/dl), LDL cholesterol (>, ≤ 130 mg/dl), HDL cholesterol (>, ≤ 40 mg/dl), triglycerides (>, ≤ 150 mg/dl). Significant associations between the ACE c.2306-117_404 I/D polymorphism and the Gensini score in men with high total cholesterol levels (P(Kruskal-Wallis) = 0.008; P(adjusted) = 0.009), high level of LDL cholesterol (P(Kruskal-Wallis) = 0.016; P(adjusted) = 0.028) and low level of HDL cholesterol (P(Kruskal-Wallis) = 0.04; P(adjusted) = 0.055) have been found. No association between the AGTR1 c.1080*86A>C and CYP11B2 c.-344C>T and the Gensini score has been found. These results suggest that men who carry ACE c.2306-117_404 DD genotype and have high total cholesterol, high LDL cholesterol and low HDL cholesterol levels may be predisposed to the development of more severe CAD.

Association of the aldosterone synthase gene -344T>C polymorphism with essential hypertension and glucose homeostasis: a case-control study in a Han Chinese population.

1. Aldosterone is a hormone that affects both blood pressure and glucose homeostasis. We studied the association of the aldosterone synthase gene (CYP11B2) polymorphism -344T>C with essential hypertension (EH) and glucose homeostasis in people in China. 2. We investigated the polymorphism -344T>C in CYP11B2 using a case-control study design (1059 cases and 1120 controls). Genotyping was carried out by matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy using a MassARRAY platform. 3. The aldosterone synthase gene -344T>C polymorphism was found to be associated with EH (odds ratio 1.252; 95% confidence interval 1.067-1.468; P(add) = 0.006). The -344C variant was found to be significantly associated with increased systolic blood pressure (P(add) = 0.003) and diastolic blood pressure (P(add) = 0.024) in controls and increased plasma aldosterone levels (P(add) = 0.0001) in EH cases. The -344C variant was also found to be significantly associated with increased fasting glucose (P(rec) = 0.003) in controls. In the subgroup containing 893 EH cases without a history of diabetes or hypoglycaemia medications, the -344C variant was found to be associated with increased fasting and postprandial plasma glucose levels (P(add) = 0.0001, P(add) = 0.001, respectively) and decreased pancreatic ß-cell function and insulin sensitivity (P(add) = 0.030, P(dom) = 0.019, respectively) by homeostasis model assessments. 4. In this Han Chinese population, the -344T/C polymorphism of the CYP11B2 gene was found to be associated with EH and glucose homeostasis, both of which might be mediated by plasma aldosterone levels, insulin sensitivity and ß-cell function.

[Comparative genetic analysis of different forms of low-renin arterial hypertension].

High level of clinical and genetic heterogeneity is a characteristic of arterial hypertension (AH) that is one of the most wide-spread cardiovascular diseases. In most cases (excluding a few monogenic forms), AH is a polygenic disease and genes of renin-angiotensin-aldosterone system play an important role in AH predisposition. 20-25% AH cases occur during low activity of renin in blood plasma (low-renin form of AH) while aldosterone production can be increased (hyperaldosteronism, HA) or normal. We examined polymorphism of genes that code the renin-angiotensin-aldosterone system components in the groups of low-renin forms of AH, namely, primary HA, idiopathic HA and AH with normal level of aldosterone. For all HA cases, the absence of chimeric CYP11B2/CYP11B1 gene that is a cause for monogenic disease--amilial HA of first type, was shown. A comparison of distributions of alleles and genotypes of polymorphous regions of genes: CYP11B2 (C-344T), REN (C-5434T, C-5312T and A BglI G), AGT (Thr174Met), ACE (I/D), CMA (G-1903A), AT2R1 (A1166C) and of their combinations is the groups described above was done. The analysis of carriership of the alleles and genotypes combinations of the polymorphous regions has shown that genes CYP11B2, REN, ACE, CMA andA T2R1 participate in development of low-renin HA. The results are evidence of similarities and some definite differences in genetic nature of the different forms of low-renin AH and, to say more widely, argue that the investigation of genetic predisposition for clinically heterogeneous forms of polygene diseases by comparison of groups of patients, separated in accordance with peculiarities of disease course, holds much promise for their hereditary background understanding.

[Contribution of CYP11B2, REN and AGT genes in genetic predisposition to arterial hypertension associated with hyperaldosteronism].

We carried out comparison of distribution of alleles and genotypes of polymorphic loci of renin-angiotensin-aldosterone system genes: CYP11B2 (C-344T), AGT (Thr174Met) and REN (C-5434T, C-5312T, and A BglI G) and their combinations in two groups of patients with low renin forms of arterial hypertension (AH). Group 1 included 59 patients with low renin hyperaldosteronism (HA) at the background of glomerular zone adenoma and hyperplasia of adrenal cortex. Group 2 included 28 patients with low renin hypertensive disease characterized by normal level of aldosterone. Complex analysis of carriership of allele and genotype combinations evidence for the difference in genetic nature of two forms of low renin AH. Participation of CYP11B2 and REN and possibly AGT genes in development of low renin AH was convincingly shown.

Testicular tumors in patients with congenital adrenal hyperplasia due to 21-hydroxylase deficiency show functional features of adrenocortical tissue.

CONTEXT: In male patients with congenital adrenal hyperplasia (CAH), testicular adrenal rest tumors are frequently found that may interfere with gonadal function. OBJECTIVE: Our objective was to determine steroid-producing features of testicular adrenal rest tumors. DESIGN AND SETTING: The study is descriptive and took place at a university medical center. PATIENTS: Eight adult CAH patients with bilateral testicular adrenal rest tumors were treated with testis-sparing surgery. INTERVENTIONS: In all but one patient, spermatic veins were cannulated during surgery and blood samples collected to measure the adrenal-specific steroid 21-deoxycortisol (21DF) and 17-hydroxyprogesterone (17OHP) and androstenedione (A). The same parameters were measured in simultaneously taken peripheral blood. mRNA concentrations of adrenal-specific enzymes CYP11B1 and CYP11B2 and ACTH and angiotensin II (AII) receptors were measured in tumor tissue. MAIN OUTCOME MEASURES: Adrenal-specific steroids/enzymes were assessed. RESULTS: 21DF, 17OHP, and A levels were measurable in all spermatic vein samples. The ratio (mean +/- SD) between spermatic vein and simultaneously taken peripheral blood samples was 37.8 +/- 56.3 (21DF), 132.0 +/- 249 (17OHP), and 57.0 +/- 68.2 (A). CYP11B1, CYP11B2, and ACTH and AII receptor mRNAs were detected in all tumors with a strong correlation between ACTH receptor mRNA in tumors and 21DF (r = 0.85; P = 0.015), 17OHP (r = 1; P = 0.01) and A (r = 0.89; P = 0.007) concentrations in peripheral blood. CONCLUSION: Testicular adrenal rest tumors produce adrenal-specific steroids and express adrenal-specific enzymes and ACTH and AII receptors, confirming the strong resemblance with adrenal tissue. Because AII receptors are present in tumor tissue, it can be hypothesized that AII may be an additional factor responsible for testicular adrenal rest tumor growth.

Aldosterone synthase -344C/T and angiotensin-converting enzyme I/D polymorphisms in Turkish hypertensive patients with normal coronary arteries.

BACKGROUND: The renin-angiotensin-aldosterone system (RAAS) plays an important role in blood pressure regulation, left ventricular and vascular structure. Some hypertensive patients develop left ventricular dilatation and heart failure symptoms while others are relatively less affected. The purpose of our study was to determine whether two major polymorphisms of RAAS are associated with echocardiographic left ventricular mass, function and dilatation in hypertensive patients with normal coronary arteries. METHODS: A Turkish population of 88 patients with hypertension and normal coronary arteries were studied by echocardiography for left ventricular dimension, mass and function. Genotyping was performed for aldosterone synthase (CYP11B2) -344C/T polymorphism in 85, angiotensin-converting enzyme (ACE) I/D polymorphism in 88 patients. Left ventricular dimensions, mass and function indices, after adjustment for clinical covariates, were analysed by multiple regression according to genotypes. RESULTS: None of the two genotypes studied predicted left ventricular dilatation, mass or function in hypertensive patients with normal coronary arteries. CONCLUSION: Neither ACE I/D nor CYP11B2 -344C/T polymorphisms were useful to predict left ventricular mass, function or dilatation in our hypertensive patients with normal coronary arteries.

Renin-angiotensin-aldosterone system in brain infarction and vascular death.

The renin-angiotensin-aldosterone system has functions that may contribute to brain infarction (BI). In 459 matched pairs of white patients and control subjects, we measured plasma angiotensin-converting enzyme (ACE) levels, seven polymorphisms (angiotensinogen T174M and M235T, ACE I/D and 4656 2/3CT repeat [rpt], angiotensin II type 1 receptor A1166C and A153G, and aldosterone synthase CYP11B2), and evaluated 5-year poststroke mortality. Mean plasma ACE levels (+/-standard error) were significantly greater in patients than control subjects (37.5 +/- 0.9 vs 33.9 +/- 0.9), in patients with lacunar stroke, and in patients with no previous vascular (cerebrovascular or cardiovascular) history. The risk for BI increased with tertiles of plasma ACE, without an interaction with hypertension. After adjustments, the association disappeared except among patients with cardioembolic BI and those without previous vascular events. Among the polymorphisms, there was a weak association of BI with angiotensin II type 1 receptor 1166C, a weak protective effect with angiotensinogen 174M, and a strong association of angiotensinogen 235T with 5-year vascular mortality. These results suggest that renin-angiotensin-aldosterone system activity and genes contribute to cerebrovascular disease and poststroke vascular death in white patients.