Whole blood methylome-derived features to discriminate endocrine hypertension.

BACKGROUND: Arterial hypertension represents a worldwide health burden and a major risk factor for cardiovascular morbidity and mortality. Hypertension can be primary (primary hypertension, PHT), or secondary to endocrine disorders (endocrine hypertension, EHT), such as Cushing's syndrome (CS), primary aldosteronism (PA), and pheochromocytoma/paraganglioma (PPGL). Diagnosis of EHT is currently based on hormone assays. Efficient detection remains challenging, but is crucial to properly orientate patients for diagnostic confirmation and specific treatment. More accurate biomarkers would help in the diagnostic pathway. We hypothesized that each type of endocrine hypertension could be associated with a specific blood DNA methylation signature, which could be used for disease discrimination. To identify such markers, we aimed at exploring the methylome profiles in a cohort of 255 patients with hypertension, either PHT (n = 42) or EHT (n = 213), and at identifying specific discriminating signatures using machine learning approaches. RESULTS: Unsupervised classification of samples showed discrimination of PHT from EHT. CS patients clustered separately from all other patients, whereas PA and PPGL showed an overall overlap. Global methylation was decreased in the CS group compared to PHT. Supervised comparison with PHT identified differentially methylated CpG sites for each type of endocrine hypertension, showing a diffuse genomic location. Among the most differentially methylated genes, FKBP5 was identified in the CS group. Using four different machine learning methods-Lasso (Least Absolute Shrinkage and Selection Operator), Logistic Regression, Random Forest, and Support Vector Machine-predictive models for each type of endocrine hypertension were built on training cohorts (80% of samples for each hypertension type) and estimated on validation cohorts (20% of samples for each hypertension type). Balanced accuracies ranged from 0.55 to 0.74 for predicting EHT, 0.85 to 0.95 for predicting CS, 0.66 to 0.88 for predicting PA, and 0.70 to 0.83 for predicting PPGL. CONCLUSIONS: The blood DNA methylome can discriminate endocrine hypertension, with methylation signatures for each type of endocrine disorder.

Preanalytical Pitfalls in Untargeted Plasma Nuclear Magnetic Resonance Metabolomics of Endocrine Hypertension.

Despite considerable morbidity and mortality, numerous cases of endocrine hypertension (EHT) forms, including primary aldosteronism (PA), pheochromocytoma and functional paraganglioma (PPGL), and Cushing's syndrome (CS), remain undetected. We aimed to establish signatures for the different forms of EHT, investigate potentially confounding effects and establish unbiased disease biomarkers. Plasma samples were obtained from 13 biobanks across seven countries and analyzed using untargeted NMR metabolomics. We compared unstratified samples of 106 PHT patients to 231 EHT patients, including 104 PA, 94 PPGL and 33 CS patients. Spectra were subjected to a multivariate statistical comparison of PHT to EHT forms and the associated signatures were obtained. Three approaches were applied to investigate and correct confounding effects. Though we found signatures that could separate PHT from EHT forms, there were also key similarities with the signatures of sample center of origin and sample age. The study design restricted the applicability of the corrections employed. With the samples that were available, no biomarkers for PHT vs. EHT could be identified. The complexity of the confounding effects, evidenced by their robustness to correction approaches, highlighted the need for a consensus on how to deal with variabilities probably attributed to preanalytical factors in retrospective, multicenter metabolomics studies.

Prediction of hyperaldosteronism subtypes when adrenal vein sampling is unilaterally successful.

OBJECTIVE: Adrenal venous sampling (AVS) is the gold standard to discriminate patients with unilateral primary aldosteronism (UPA) from bilateral disease (BPA). AVS is technically demanding and in cases of unsuccessful cannulation of adrenal veins, the results may not always be interpreted. The aim of our study was to develop diagnostic models to distinguish UPA from BPA, in cases of unilateral successful AVS and the presence of contralateral suppression of aldosterone secretion. DESIGN: Retrospective evaluation of 158 patients referred to a tertiary hypertension unit who underwent AVS. We randomly assigned 110 patients to a training cohort and 48 patients to a validation cohort to develop and test the diagnostic models. METHODS: Supervised machine learning algorithms and regression models were used to develop and validate two prediction models and a simple 19-point score system to stratify patients according to their subtype diagnosis. RESULTS: Aldosterone levels at screening and after confirmatory testing, lowest potassium, ipsilateral and contralateral imaging findings at CT scanning, and contralateral ratio at AVS, were associated with a diagnosis of UPA and were included in the diagnostic models. Machine learning algorithms correctly classified the majority of patients both at training and validation (accuracy: 82.9-95.7%). The score system displayed a sensitivity/specificity of 95.2/96.9%, with an AUC of 0.971. A flow-chart integrating our score correctly managed all patients except 3 (98.1% accuracy), avoiding the potential repetition of 77.2% of AVS procedures. CONCLUSIONS: Our score could be integrated in clinical practice and guide surgical decision-making in patients with unilateral successful AVS and contralateral suppression.

Patients With Primary Aldosteronism Respond to Unilateral Adrenalectomy With Long-Term Reduction in Salt Intake.

CONTEXT: High dietary salt intake is known to aggravate arterial hypertension. This effect could be of particular relevance in the setting of primary aldosteronism (PA), which is associated with cardiovascular damage independent of blood pressure levels. The aim of this study was to determine the impact of therapy on salt intake in PA patients. PATIENTS AND METHODS: A total of 148 consecutive PA patients (66 with unilateral and 82 with bilateral PA) from the database of the German Conn's Registry were included. Salt intake was quantified by 24-hour urinary sodium excretion before and after initiation of PA treatment. STUDY DESIGN: Observational longitudinal cohort study. SETTING: Tertiary care hospital. RESULTS: At baseline, unilateral PA patients had a significantly higher urinary sodium excretion than patients with bilateral disease (205 vs 178 mmol/d, P = 0.047). Higher urinary sodium excretion correlated with an increased cardiovascular risk profile including proteinuria, impaired lipid, and glucose metabolism and was associated with higher daily doses of antihypertensive drugs to achieve blood pressure control. In unilateral disease, urinary sodium excretion dropped spontaneously to 176 mmol/d (P = 0.012) 1 year after unilateral adrenalectomy and remained low at 3 years of follow-up (174 mmol/d). In contrast, treatment with mineralocorticoid receptor antagonists (MRA) in bilateral PA patients was not associated with a significant change in urinary sodium excretion at follow-up (179 mmol/d vs 183 mmol/d). CONCLUSION: PA patients consuming a high-salt diet, estimated based on urinary sodium excretion, respond to adrenalectomy with a significant reduction of salt intake, in contrast to MRA treatment.

Computed Tomography and Adrenal Venous Sampling in the Diagnosis of Unilateral Primary Aldosteronism.

Unilateral primary aldosteronism is the most common surgically correctable form of endocrine hypertension and is usually differentiated from bilateral forms by adrenal venous sampling (AVS) or computed tomography (CT). Our objective was to compare clinical and biochemical postsurgical outcomes of patients with unilateral primary aldosteronism diagnosed by CT or AVS and identify predictors of surgical outcomes. Patient data were obtained from 18 internationally distributed centers and retrospectively analyzed for clinical and biochemical outcomes of adrenalectomy of patients with surgical management based on CT (n=235 patients, diagnosed from 1994-2016) or AVS (526 patients, diagnosed from 1994-2015) using the standardized PASO (Primary Aldosteronism Surgical Outcome) criteria. Biochemical outcomes were highly different according to surgical management approach with a smaller proportion in the CT group achieving complete biochemical success (188 of 235 [80%] patients versus 491 of 526 [93%], P<0.001) and a greater proportion with absent biochemical success (29 of 235 [12%] versus 10 of 526 [2%], P<0.001). A diagnosis by CT was associated with a decreased likelihood of complete biochemical success compared with AVS (odds ratio, 0.28; 0.16-0.50; P<0.001). Clinical outcomes were not significantly different, but the absence of a postsurgical elevated aldosterone-to-renin ratio was a strong marker of complete clinical success (odds ratio, 14.81; 1.76-124.53; P=0.013) in the CT but not in the AVS group. In conclusion, patients diagnosed by CT have a decreased likelihood of achieving complete biochemical success compared with a diagnosis by AVS.

Comparative Genomics and Transcriptome Profiling in Primary Aldosteronism.

Primary aldosteronism is the most common form of endocrine hypertension with a prevalence of 6% in the general population with hypertension. The genetic basis of the four familial forms of primary aldosteronism (familial hyperaldosteronism FH types I-IV) and the majority of sporadic unilateral aldosterone-producing adenomas has now been resolved. Familial forms of hyperaldosteronism are, however, rare. The sporadic forms of the disease prevail and these are usually caused by either a unilateral aldosterone-producing adenoma or bilateral adrenal hyperplasia. Aldosterone-producing adenomas frequently carry a causative somatic mutation in either of a number of genes with the KCNJ5 gene, encoding an inwardly rectifying potassium channel, a recurrent target harboring mutations at a prevalence of more than 40% worldwide. Other than genetic variations, gene expression profiling of aldosterone-producing adenomas has shed light on the genes and intracellular signalling pathways that may play a role in the pathogenesis and pathophysiology of these tumors.

Outcomes after adrenalectomy for unilateral primary aldosteronism: an international consensus on outcome measures and analysis of remission rates in an international cohort.

BACKGROUND: Although unilateral primary aldosteronism is the most common surgically correctable cause of hypertension, no standard criteria exist to classify surgical outcomes. We aimed to create consensus criteria for clinical and biochemical outcomes and follow-up of adrenalectomy for unilateral primary aldosteronism and apply these criteria to an international cohort to analyse the frequency of remission and identify preoperative determinants of successful outcome. METHODS: The Primary Aldosteronism Surgical Outcome (PASO) study was an international project to develop consensus criteria for outcomes and follow-up of adrenalectomy for unilateral primary aldosteronism. An international panel of 31 experts from 28 centres, including six endocrine surgeons, used the Delphi method to reach consensus. We then retrospectively analysed follow-up data from prospective cohorts for outcome assessment of patients diagnosed with unilateral primary aldosteronism by adrenal venous sampling who had undergone a total adrenalectomy, consecutively included from 12 referral centres in nine countries. On the basis of standardised criteria, we determined the proportions of patients achieving complete, partial, or absent clinical and biochemical success in accordance with the consensus. We then used logistic regression analyses to identify preoperative factors associated with clinical and biochemical outcomes. FINDINGS: Consensus was reached for criteria for six outcomes (complete, partial, and absent success of clinical and biochemical outcomes) based on blood pressure, use of antihypertensive drugs, plasma potassium and aldosterone concentrations, and plasma renin concentrations or activities. Consensus was also reached for two recommendations for the timing of follow-up assessment. For the international cohort analysis, we analysed clinical data from 705 patients recruited between 1994 and 2015, of whom 699 also had biochemical data. Complete clinical success was achieved in 259 (37%) of 705 patients, with a wide variance (range 17-62), and partial clinical success in an additional 334 (47%, range 35-66); complete biochemical success was seen in 656 (94%, 83-100) of 699 patients. Female patients had a higher likelihood of complete clinical success (odds ratio [OR] 2·25, 95% CI 1·40-3·62; p=0·001) and clinical benefit (complete plus partial clinical success; OR 2·89, 1·49-5·59; p=0·002) than male patients. Younger patients had a higher likelihood of complete clinical success (OR 0·95 per extra year, 0·93-0·98; p<0·001) and clinical benefit (OR 0·95 per extra year, 0·92-0·98; p=0·004). Higher levels of preoperative medication were associated with lower levels of complete clinical success (OR 0·80 per unit increase, 0·70-0·90; p<0·001). INTERPRETATION: These standardised outcome criteria are relevant for the assessment of the success of surgical treatment in individual patients and will allow the comparison of outcome data in future studies. The variable baseline clinical characteristics of our international cohort contributed to wide variation in clinical outcomes. Most patients derive clinical benefit from adrenalectomy, with younger patients and female patients more likely to have a favourable surgical outcome. Screening for primary aldosteronism should nonetheless be done in every individual fulfilling US Endocrine Society guideline criteria because biochemical success without clinical success is by itself clinically important and older women and men can also derive post-operative clinical benefit. FUNDING: European Research Council; European Union's Horizon 2020; Else Kröner-Fresenius Stiftung; Netherlands Organisation for Health Research and Development-Medical Sciences; Japanese Ministry of Health, Labour and Welfare; Ministry of Health, Slovenia; US National Institutes of Health; and CONICYT-FONDECYT (Chile).

Is There a Role for Genomics in the Management of Hypertension?

Hypertension (HTN) affects about 1 billion people worldwide and the lack of a single identifiable cause complicates its treatment. Blood pressure (BP) levels are influenced by environmental factors, but there is a strong genetic component. Linkage analysis has identified several genes involved in Mendelian forms of HTN and the associated pathophysiological mechanisms have been unravelled, leading to targeted therapies. The majority of these syndromes are due to gain-of-function or loss-of-functions mutations, resulting in an alteration of mineralocorticoid, glucocorticoid, or sympathetic pathways. The diagnosis of monogenic forms of HTN has limited practical implications on the population and a systematic genetic screening is not justifiable. Genome-wide linkage and association studies (GWAS) have identified single nucleotide polymorphisms (SNPs), which influence BP. Forty-three variants have been described with each SNP affecting systolic and diastolic BP by 1.0 and 0.5 mmHg, respectively. Taken together Mendelian inheritance and all GWAS-identified HTN-associated variants explain 2-3% of BP variance. Epigenetic modifications, such as DNA methylation, histone modification and non-coding RNAs, have become increasingly recognized as important players in BP regulation and may justify a further part of missing heritability. In this review, we will discuss how genetics and genomics may assist clinicians in managing patients with HTN.

Reference intervals for plasma concentrations of adrenal steroids measured by LC-MS/MS: Impact of gender, age, oral contraceptives, body mass index and blood pressure status.

BACKGROUND: Mass spectrometric-based measurements of the steroid metabolome have been introduced to diagnose disorders featuring abnormal steroidogenesis. Defined reference intervals are important for interpreting such data. METHODS: Liquid chromatography-tandem mass spectrometry was used to establish reference intervals for 16 steroids (pregnenolone, progesterone, 11-deoxycorticosterone, corticosterone, aldosterone, 18-oxocortisol, 18-hydroxycortisol, 17-hydroxyprogesterone, 21-deoxycortisol, 11-deoxycortisol, cortisol, cortisone, dehydroepiandrosterone, dehydroepiandrosterone-sulfate, androstenedione, testosterone) measured in plasma from 525 volunteers with (n=227) and without (n=298) hypertension, including 68 women on oral contraceptives. RESULTS: Women showed variable plasma concentrations of several steroids associated with menstrual cycle phase, menopause and oral contraceptive use. Progesterone was higher in females than males, but most other steroids were higher in males than females and almost all declined with advancing age. Using models that corrected for age and gender, body mass index showed weak negative relationships with corticosterone, 21-deoxycortisol, cortisol, cortisone, testosterone, progesterone, 17-hydroxyprogesterone and 11-deoxycorticosterone, but a positive relationship with 18-hydroxycortisol. Hypertensives and normotensives showed negligible differences in plasma concentrations of steroids. CONCLUSION: Age and gender are the most important variables for plasma steroid reference intervals, which have been established here according to those variables for a panel of 16 steroids primarily useful for diagnosis and subtyping of patients with endocrine hypertension.

Prevalence and Clinical Manifestations of Primary Aldosteronism Encountered in Primary Care Practice.

BACKGROUND: Despite being widely recognized as the most common form of secondary hypertension, among the general hypertensive population the true prevalence of primary aldosteronism (PA) and its main subtypes, aldosterone-producing adenoma (APA) and bilateral adrenal hyperplasia (BAH), remains a matter of debate. OBJECTIVES: This study sought to determine the prevalence and clinical phenotype of PA in a large cohort of unselected patients with hypertension, consecutively referred to our hypertension unit, by 19 general practitioners from Torino, Italy. METHODS: Following withdrawal from all interfering medications, patients were screened for PA using the ratio of serum aldosterone to plasma renin activity. PA was diagnosed according to Endocrine Society guidelines. The diagnosis was confirmed or excluded by an intravenous saline infusion test or captopril challenge test and subtype differentiation was performed by adrenal computed tomography scanning and adrenal vein sampling, using strict criteria to define successful cannulation and lateralization of aldosterone production. RESULTS: A total of 1,672 primary care patients with hypertension (569 newly diagnosed and 1,103 patients already diagnosed with arterial hypertension) were included in the study. A total of 99 patients (5.9%) were diagnosed with PA and conclusive subtype differentiation by adrenal vein sampling was made in 91 patients (27 patients with an APA and 64 patients with BAH). The overall prevalence of PA increased with the severity of hypertension, from 3.9% in stage 1 hypertension to 11.8% in stage 3 hypertension. Patients with PA more frequently displayed target organ damage and cardiovascular events compared with those without PA, independent of confounding variables. CONCLUSIONS: Our results demonstrated that PA is a frequent cause of secondary hypertension, even in the general population of patients with hypertension, and indicates that most of these patients should be screened for PA.

Subtype Diagnosis of Primary Aldosteronism: Is Adrenal Vein Sampling Always Necessary?

Aldosterone producing adenoma and bilateral adrenal hyperplasia are the two most common subtypes of primary aldosteronism (PA) that require targeted and distinct therapeutic approaches: unilateral adrenalectomy or lifelong medical therapy with mineralocorticoid receptor antagonists. According to the 2016 Endocrine Society Guideline, adrenal venous sampling (AVS) is the gold standard test to distinguish between unilateral and bilateral aldosterone overproduction and therefore, to safely refer patients with PA to surgery. Despite significant advances in the optimization of the AVS procedure and the interpretation of hormonal data, a standardized protocol across centers is still lacking. Alternative methods are sought to either localize an aldosterone producing adenoma or to predict the presence of unilateral disease and thereby substantially reduce the number of patients with PA who proceed to AVS. In this review, we summarize the recent advances in subtyping PA for the diagnosis of unilateral and bilateral disease. We focus on the developments in the AVS procedure, the interpretation criteria, and comparisons of the performance of AVS with the alternative methods that are currently available.

Guidelines for primary aldosteronism: uptake by primary care physicians in Europe.

CONTEXT: Primary aldosteronism is the most frequent form of secondary hypertension but is still markedly underdiagnosed. In 2008, the Endocrine Society released guidelines for primary aldosteronism diagnosis and management; current indications are that they are not being widely followed. OBJECTIVE: To investigate the level of knowledge and application of the guidelines in a large representative cohort of general practitioners (GPs) in Italy and Germany. SETTING AND DESIGN: The study was carried out by web questionnaire on hypertension and primary aldosteronism management with 500 GPs (250 in Italy and 250 in Germany), stratified by geographical area and city size. RESULTS: The mean number of patients seen was 1747 (Germany) and 1388 (Italy). Of these, 18% were diagnosed as hypertensive in Italy and 25% in Germany. Renin and aldosterone measurements were ordered by 7% of GPs in Italy and 8% in Germany. GPs in Italy considered 8% of patients eligible for aldosterone and renin measurements compared with 13% of GPs in Germany. In Italy, primary aldosteronism prevalence was 1% among hypertensive patients; 36% of the GPs reported no primary aldosteronism patients under their care or diagnosed previously. In Germany, the prevalence of primary aldosteronism was 2% among hypertensive patients; 19% of GPs had no primary aldosteronism patients. CONCLUSION: In Germany and in Italy, primary aldosteronism is not widely recognized by GPs; Endocrine Society guidelines for primary aldosteronism diagnosis are not well known or applied, resulting in marked underdiagnosis of the disease.

Diagnostic accuracy of aldosterone and renin measurement by chemiluminescent immunoassay and radioimmunoassay in primary aldosteronism.

OBJECTIVE: Up to 50% of hypertensive patients should be screened for primary aldosteronism, using the aldosterone to renin (or plasma renin activity) ratio [aldosterone to active renin ratio (AARR) and aldosterone to plasma renin activity ratio (ARR), respectively]. Aim of the study was to prospectively compare the diagnostic accuracy of AARR (measured by chemiluminescent immunoassay) and ARR (measured by radioimmunoassay) as screening tests for primary aldosteronism and aldosterone assays (measured by chemiluminescence and radioimmunoassay) during confirmatory testing. METHODS: One hundred patients were screened for primary aldosteronism and 34 underwent confirmatory testing. The cut-offs for ARR and AARR were 30 ng/dl/ng/ml/h and 3.7 ng/dl/mU/l, respectively. Patients with positive confirmatory test underwent subtype diagnosis. RESULTS: Seventy-five patients were essential hypertensive patients, 15 had idiopathic hyperaldosteronism, five aldosterone-producing adenoma (APA) and five with undefined diagnosis. The AARR displayed a sensitivity of 90% and a specificity of 99%, the ARR had a sensitivity of 100% and a specificity of 73%. Of the two of 20 primary aldosteronism patients missed by AARR, none resulted affected by APA. All primary aldosteronism patients were correctly diagnosed by chemiluminescence at confirmatory testing. In the total sample of 168 measurements both the correlation for plasma renin activity with renin and for aldosterone in chemiluminescence and radioimmunoassay were highly significant (ρ = 0.70, P < 0.001 and ρ = 0.78, P < 0.001, respectively). On receiver operator characteristics curves, the area under the curve for AARR was 0.989 [95% confidence interval (CI) 0.97-1] and 0.934 for ARR (95% CI 0.89-0.98), which were not significantly different. CONCLUSION: The automated aldosterone and renin chemiluminescent assay is a reliable alternative to the radioimmunometric method, especially for APA detection.

Mass Spectrometry-Based Adrenal and Peripheral Venous Steroid Profiling for Subtyping Primary Aldosteronism.

BACKGROUND: Differentiating patients with primary aldosteronism caused by aldosterone-producing adenomas (APAs) from those with bilateral adrenal hyperplasia (BAH), which is essential for choice of therapeutic intervention, relies on adrenal venous sampling (AVS)-based measurements of aldosterone and cortisol. We assessed the utility of LC-MS/MS-based steroid profiling to stratify patients with primary aldosteronism. METHODS: Fifteen adrenal steroids were measured by LC-MS/MS in peripheral and adrenal venous plasma from AVS studies for 216 patients with primary aldosteronism at 3 tertiary referral centers. Ninety patients were diagnosed with BAH and 126 with APAs on the basis of immunoassay-derived adrenal venous aldosterone lateralization ratios. RESULTS: Among 119 patients confirmed to have APAs at follow-up, LC-MS/MS-derived lateralization ratios of aldosterone normalized to cortisol, dehydroepiandrosterone, and androstenedione were all higher (P < 0.0001) than immunoassay-derived ratios. The hybrid steroids, 18-oxocortisol and 18-hydroxycortisol, also showed lateralized secretion in 76% and 35% of patients with APAs. Adrenal venous concentrations of glucocorticoids and androgens were bilaterally higher in patients with BAH than in those with APAs. Consequently, peripheral plasma concentrations of 18-oxocortisol were 8.5-fold higher, whereas concentrations of cortisol, corticosterone, and dehydroepiandrosterone were lower in patients with APAs than in those with BAH. Correct classification of 80% of cases of APAs vs BAH was thereby possible by use of a combination of steroids in peripheral plasma. CONCLUSIONS: LC-MS/MS-based steroid profiling during AVS achieves higher aldosterone lateralization ratios in patients with APAs than immunoassay. LC-MS/MS also enables multiple measures for discriminating unilateral from bilateral aldosterone excess, with potential use of peripheral plasma for subtype classification.

Immunohistochemical, genetic and clinical characterization of sporadic aldosterone-producing adenomas.

Adrenal glands removed for unilateral primary aldosteronism (PA) display marked histological heterogeneity. Recently reported somatic mutations in KCNJ5, ATP1A1, ATP2B3 and CACNA1D can partially account for these differences. In this study we aimed at combining phenotypic and genotypic characteristics, integrating genetic and immunohistochemistry correlates in sporadic PA. Seventy-one adrenal glands have been included in the study and analyzed for mutations in KCNJ5, ATP1A1, ATP2B3 and CACNA1D. Histological examination and immunohistochemical staining for CYP11B1 (11ß-hydroxylase) and CYP11B2 (aldosterone synthase) were performed on aldosterone-producing adenomas (APAs) and adjacent adrenal cortex. In our cohort, the final histopathological diagnosis was multinodular hyperplasia in 22.5% of the patients and single nodule in 77.5%. Forty-five percent of the removed adrenals displayed extra-APA CYP11B2-positive cell nests (B2-CN). Among adrenal vein sampling parameters the suppression of contralateral adrenal was more frequent and the lateralization index was higher in the subgroup of patients without extra-APA B2-CN compared to the subgroup with extra-APA B2-CN. KCNJ5-mutated APAs were composed mainly of zona fasciculata-like cells with high expression of CYP11B1, while ATP1A1, ATP2B3 and CACNA1D-mutated APAs presented more frequently a zona-glomerulosa-like phenotype with high expression of CYP11B2. We observed a significant inverse correlation between CYP11B2 expression and the size of the nodules and, if CYP11B2 expression was corrected for tumor volume, a significant correlation with plasma aldosterone and aldosterone to renin ratio. Our findings indicate that combination of genotyping and immunohistochemistry improves the final histopathological diagnosis between single nodule and multinodular hyperplasia of the assessed adrenals.

Understanding primary aldosteronism: impact of next generation sequencing and expression profiling.

Primary aldosteronism (PA) encompasses a broad, heterogeneous group of disorders including both sporadic and familial forms (familial hyperaldosteronism type I, II and III). PA is the most common form of secondary hypertension and associated with a higher rate of cardiovascular complications, compared with essential hypertension. Despite significant progress in the diagnosis and management of PA, until recently the molecular mechanisms leading to inappropriate aldosterone production were largely unknown. The introduction of next-generation sequencing has had a profound impact on the field of human genetics and has given new insight in the molecular determinants that lead to both sporadic and familial forms of PA. Here we review the recent progress toward understanding of the genetic and molecular mechanisms leading to autonomous aldosterone production in PA.

A case of severe hyperaldosteronism caused by a de novo mutation affecting a critical salt bridge Kir3.4 residue.

CONTEXT: Familial hyperaldosteronism type III (FH-III) is a rare and clinically heterogeneous condition, that can display mild as well as severe phenotypes. Point mutations in the KCNJ5 gene, affecting the ion selectivity of the inward rectifier K(+) channel 4 (Kir3.4), underlie the molecular basis of FH-III. OBJECTIVE: The objective of the study was to investigate the effects of a de novo germline KCNJ5 mutation. PATIENTS AND METHODS: We describe the case of a girl who came to medical attention at the age of 2 years because of polydipsia, polyuria, and failure to thrive. The patient, affected by hypertension and hypokalemia, was diagnosed with primary aldosteronism on the basis of extremely high aldosterone levels and suppressed plasma renin activity. Genomic DNA was isolated and KCNJ5 sequenced. Human adrenocortical cells were used as an in vitro model for the functional characterization of the mutant channel. RESULTS: KCNJ5 sequencing in the index case and her parents revealed a de novo p.Glu145Gln germline mutation. The substitution resulted in Na(+)-dependent depolarization of adrenal cells and increased intracellular calcium concentration, which activated the transcription of NR4A2 and, in turn, CYP11B2. Pharmacological studies revealed that the mutant channel was insensitive to tertiapin-Q and calcium-channel blocker verapamil. CONCLUSIONS: Herein we report the identification of a novel KCNJ5 germline mutation responsible for severe hyperaldosteronism that presented in infancy with symptoms of diabetes insipidus. The findings of this study further elucidate the etiology of FH-III and expand our knowledge of this rare condition.

Genome-wide association study identifies CAMKID variants involved in blood pressure response to losartan: the SOPHIA study.

BACKGROUND: Essential hypertension arises from the combined effect of genetic and environmental factors. A pharmacogenomics approach could help to identify additional molecular mechanisms involved in its pathogenesis. AIM: The aim of SOPHIA study was to identify genetic polymorphisms regulating blood pressure response to the angiotensin II receptor blocker, losartan, with a whole-genome approach. MATERIALS & METHODS: We performed a genome-wide association study on blood pressure response in 372 hypertensives treated with losartan and we looked for replication in two independent samples. RESULTS: We identified a peak of association in CAMK1D gene (rs10752271, effect size -5.5 ± 0.94 mmHg, p = 1.2 × 10(-8)). CAMK1D encodes a protein that belongs to the regulatory pathway involved in aldosterone synthesis. We tested the specificity of rs10752271 for losartan in hypertensives treated with hydrochlorothiazide and we validated it in silico in the GENRES cohort. CONCLUSION: Using a genome-wide approach, we identified the CAMK1D gene as a novel locus associated with blood pressure response to losartan. CAMK1D gene characterization may represent a useful tool to personalize the treatment of essential hypertension.