Paolo Mulatero

Drug Effects on Aldosterone/Plasma Renin Activity Ratio in Primary Aldosteronism

Primary aldosteronism is a specifically treatable and potentially curable form of secondary hypertension. The aldosterone/plasma renin activity ratio (ARR) is routinely used as a screening test. An...

Somatic mutations in ATP1A1 and ATP2B3 lead to aldosterone-producing adenomas and secondary hypertension

Primary aldosteronism is the most prevalent form of secondary hypertension. To explore molecular mechanisms of autonomous aldosterone secretion, we performed exome sequencing of aldosterone-producing adenomas (APAs). We identified somatic hotspot mutations in the ATP1A1 (encoding an Na+/K+ ATPase α subunit) and ATP2B3 (encoding a Ca2+ ATPase) genes in three and two of the nine APAs, respectively. These ATPases are expressed in adrenal cells and control sodium, potassium and calcium ion homeostasis. Functional in vitro studies of ATP1A1 mutants showed loss of pump activity and strongly reduced affinity for potassium. Electrophysiological ex vivo studies on primary adrenal adenoma cells provided further evidence for inappropriate depolarization of cells with ATPase alterations. In a collection of 308 APAs, we found 16 (5.2%) somatic mutations in ATP1A1 and 5 (1.6%) in ATP2B3. Mutation-positive cases showed male dominance, increased plasma aldosterone concentrations and lower potassium concentrations compared with mutation-negative cases. In summary, dominant somatic alterations in two members of the ATPase gene family result in autonomous aldosterone secretion.

Prevalence, Clinical, and Molecular Correlates of KCNJ5 Mutations in Primary Aldosteronism

Primary aldosteronism is the most common form of secondary hypertension. Mutations in the KCNJ5 gene have been described recently in aldosterone-producing adenomas (APAs). The aim of this study was to investigate the prevalence of KCNJ5 mutations in unselected patients with primary aldosteronism and their clinical, biological and molecular correlates. KCNJ5 sequencing was performed on somatic (APA, n=380) and peripheral (APA, n=344; bilateral adrenal hyperplasia, n=174) DNA of patients with primary aldosteronism, collected through the European Network for the Study of Adrenal Tumors. Transcriptome analysis was performed in 102 tumors. Somatic KCNJ5 mutations (p.Gly151Arg or p.Leu168Arg) were found in 34% (129 of 380) of APA. They were significantly more prevalent in females (49%) than males (19%; P<10−3) and in younger patients (42.1±1.0 versus 47.6±0.7 years; P<10−3) and were associated with higher preoperative aldosterone levels (455±26 versus 376±17 ng/L; P=0.012) but not with therapeutic outcome after surgery. Germline KCNJ5 mutations were found neither in patients with APA nor those with bilateral adrenal hyperplasia. Somatic KCNJ5 mutations were specific for APA, because they were not identified in 25 peritumoral adrenal tissues or 16 cortisol-producing adenomas. Hierarchical clustering of transcriptome profiles showed that APAs with p.Gly151Arg or p.Leu168Arg mutations were indistinguishable from tumors without KCNJ5 mutations. In conclusion, although a large proportion of sporadic APAs harbors somatic KCNJ5 mutations, germline mutations are not similarly causative for bilateral adrenal hyperplasia. KCNJ5 mutation carriers are more likely to be females; younger age and higher aldosterone levels at diagnosis suggest that KCNJ5 mutations may be associated with a more florid phenotype of primary aldosteronism.

Long-Term Cardio- and Cerebrovascular Events in Patients With Primary Aldosteronism

BACKGROUND Aldosterone plays a detrimental role on the cardiovascular system and PA patients display a higher risk of events compared with EH. OBJECTIVES The objectives of the study were to compare cardio- and cerebrovascular events in patients with primary aldosteronism (PA) and matched essential hypertension (EH). METHODS We retrospectively compared the percentage of patients experiencing events at baseline and during a median follow-up of 12 years in 270 PA patients case-control matched 1:3 with EH patients and in PA subtypes [aldosterone-producing adenoma (n = 57); bilateral adrenal hyperplasia (n = 213)] vs matched EH. RESULTS A significantly higher number of PA patients experienced cardiovascular events over the entire period of the study (22.6% vs 12.7%, P < .001). At the diagnosis of PA, a higher number of patients had experienced total events (14.1% vs 8.4% EH, P = .007); furthermore, during the follow-up period, PA patients had a higher rate of events (8.5% vs 4.3% EH, P = .008). In particular, stroke and arrhythmias were more frequent in PA patients. During the follow-up, a higher percentage of PA patients developed type 2 diabetes. Parameters that were independently associated with the occurrence of all events were age, duration of hypertension, systolic blood pressure, presence of diabetes mellitus, and PA diagnosis. After division into PA subtypes, patients with either aldosterone-producing adenoma or bilateral adrenal hyperplasia displayed a higher rate of events compared with the matched EH patients. CONCLUSIONS This study demonstrates in a large population of patients the pathogenetic role of aldosterone excess in the cardiovascular system and thus the importance of early diagnosis and targeted PA treatment.

Genetic Spectrum and Clinical Correlates of Somatic Mutations in Aldosterone-Producing Adenoma

Primary aldosteronism is the most common form of secondary hypertension. Somatic mutations in KCNJ5, ATP1A1, ATP2B3, and CACNA1D have been described in aldosterone-producing adenomas (APAs). Our aim was to investigate the prevalence of somatic mutations in these genes in unselected patients with APA (n=474), collected through the European Network for the Study of Adrenal Tumors. Correlations with clinical and biochemical parameters were first analyzed in a subset of 199 patients from a single center and then replicated in 2 additional centers. Somatic heterozygous KCNJ5 mutations were present in 38% (180/474) of APAs, whereas ATP1A1 mutations were found in 5.3% (25/474) and ATP2B3 mutations in 1.7% (8/474) of APAs. Previously reported somatic CACNA1D mutations as well as 10 novel CACNA1D mutations were identified in 44 of 474 (9.3%) APAs. There was no difference in the cellular composition of APAs or in CYP11B2, CYP11B1, KCNJ5, CACNA1D, or ATP1A1 gene expression in APAs across genotypes. Patients with KCNJ5 mutations were more frequently female, diagnosed younger, and with higher minimal plasma potassium concentrations compared with CACNA1D mutation carriers or noncarriers. CACNA1D mutations were associated with smaller adenomas. These associations were largely dependent on the population structure of the different centers. In conclusion, recurrent somatic mutations were identified in 54% of APAs. Young women with APAs are more likely to be KCNJ5 mutation carriers; identification of specific characteristics or surrogate biomarkers of mutation status may lead to targeted treatment options.

Diagnosis of primary aldosteronism: from screening to subtype differentiation

Numerous studies conducted in recent years have reported an increase in the prevalence of primary aldosteronism (PA). This increase has arisen because of changes in our screening methods used to detect PA, notably the widespread use of the ratio of plasma aldosterone concentration to plasma renin activity. A positive screening result, however, is not diagnostic and requires a confirmatory test. Strategies for screening and confirmation of PA and the techniques to identify the two main subtypes of PA--aldosterone-producing adenoma (APA) and bilateral adrenal hyperplasia (BAH)--are particularly important because hypertension in APA can be cured by adrenalectomy, whereas individuals affected with BAH can receive targeted medical treatment with mineralocorticoid receptor antagonists.

KCNJ5 Mutations in European Families With Nonglucocorticoid Remediable Familial Hyperaldosteronism

Primary aldosteronism is the most frequent cause of endocrine hypertension. Three forms of familial hyperaldosteronism (FH) have been described, named FH-I to -III. Recently, a mutation of KCNJ5 has been shown to be associated with FH-III, whereas the cause of FH-II is still unknown. In this study we searched for mutations in KCNJ5 in 46 patients from 21 families with FH, in which FH-I was excluded. We identified a new germline G151E mutation in 2 primary aldosteronism–affected subjects from an Italian family and 3 somatic mutations in aldosterone-producing adenomas, T158A described previously as a germline mutation associated with FH-III, and G151R and L168R both described as somatic mutations in aldosterone-producing adenoma. The phenotype of the family with the G151E mutation was remarkably milder compared with the previously described American family, in terms of both clinical and biochemical parameters. Furthermore, patients with somatic KCNJ5 mutations displayed a phenotype indistinguishable from that of sporadic primary aldosteronism. The functional characterization of the effects of the G151E mutation in vitro showed a profound alteration of the channel function, with loss of K+ selectivity, Na+ influx, and membrane depolarization. These alterations have been postulated to be responsible for voltage gate Ca2+ channel activation, increase in cytosolic calcium, and stimulation of aldosterone production and adrenal cell proliferation. In conclusion, we describe herein a new mutation in the KCNJ5 potassium channel associated with FH-III, responsible for marked alterations of channel function but associated with a mild clinical and hormonal phenotype.

Roles of clinical criteria, computed tomography scan, and adrenal vein sampling in differential diagnosis of primary aldosteronism subtypes.

CONTEXT In patients with primary aldosteronism (PA), it is fundamental to distinguish between subtypes that benefit from different therapies. Computed tomography (CT) scans lack sensitivity and specificity and must be followed by adrenal venous sampling (AVS). Because AVS is not widely available, a list of clinical criteria that indicate the presence of an aldosterone-producing adenoma (APA) has been suggested. OBJECTIVE AND DESIGN The objective of the study was to test the sensitivity and specificity of the last generation CT scans, test prospectively the usefulness of clinical criteria in the diagnosis of APA, and develop a flow chart to be used when AVS is not easily available. SETTING Hypertensive patients referred to our hypertension unit were included in our study. PATIENTS Seventy-one patients with confirmed PA participated in our study. INTERVENTION All patients had a CT scan and underwent AVS. MAIN OUTCOME MEASURE Final diagnosis of APA was the main measure. RESULTS A total of 44 and 56% of patients were diagnosed as having an APA and a bilateral adrenal hyperplasia (BAH), respectively. Twenty percent of patients with PA displayed hypokalemia. CT scans displayed a sensitivity of 0.87 and a specificity of 0.71. The posture test displayed a lower sensitivity and specificity (0.64 and 0.70, respectively). The distribution grades of hypertension were not significantly different between APA and BAH. Biochemical criteria of high probability of APA displayed a sensitivity of 0.32 and a specificity of 0.95. CONCLUSIONS This study underlines the central role of AVS in the subtype diagnosis of PA. The use of the clinical criteria to distinguish between APA and BAH did not display a satisfactory diagnostic power.

Impact of Different Diagnostic Criteria During Adrenal Vein Sampling on Reproducibility of Subtype Diagnosis in Patients With Primary Aldosteronism

In patients with primary aldosteronism, adrenal vein sampling (AVS) is considered the only reliable technique to distinguish between unilateral and bilateral autonomous production of aldosterone, but agreement is lacking on the best criteria indicating successful cannulation and lateralization. The objective of this study was to assess the impact of differing criteria for the successful cannulation and lateralization on the reproducibility of subtype diagnosis. Sixty-two patients with confirmed primary aldosteronism underwent AVS on 2 separate occasions, because the first was unsatisfactory. We compared the different diagnoses of primary aldosteronism subtype reached using AVS data assessed by permissive (type 1), intermediate (type 2), and strict (type 3) criteria. Although 91.1% of all of the (both first and second) AVSs were “successful” by type 1 criteria (50.8% by type 2 and 33.9% by type 3), in only 35.3% of patients was the diagnosis concordant between the first and second AVS. Type 1 criteria also led to a higher rate of diagnosis of unilateral primary aldosteronism (67.3% of successful procedures) than type 2 (36.5%) or type 3 (26.2%). There was considerable disparity in the diagnosis reached using the 3 different criteria, with concordance in only 32.2%. Using either type 1 or 2 criteria, the minimal adrenal/peripheral vein cortisol ratio necessary to obtain the same diagnosis in the first and second AVS procedures was ≥2.75. In conclusion, permissive criteria for successful cannulation and lateralization on AVS achieve poor diagnostic reproducibility and should be avoided.

Somatic ATP1A1, ATP2B3, and KCNJ5 Mutations in Aldosterone-Producing Adenomas

Aldosterone-producing adenomas (APAs) cause a sporadic form of primary aldosteronism and somatic mutations in the KCNJ5 gene, which encodes the G-protein–activated inward rectifier K+ channel 4, GIRK4, account for ≈40% of APAs. Additional somatic APA mutations were identified recently in 2 other genes, ATP1A1 and ATP2B3, encoding Na+/K+-ATPase 1 and Ca2+-ATPase 3, respectively, at a combined prevalence of 6.8%. We have screened 112 APAs for mutations in known hotspots for genetic alterations associated with primary aldosteronism. Somatic mutations in ATP1A1, ATP2B3, and KCNJ5 were present in 6.3%, 0.9%, and 39.3% of APAs, respectively, and included 2 novel mutations (Na+/K+-ATPase p.Gly99Arg and GIRK4 p.Trp126Arg). CYP11B2 gene expression was higher in APAs harboring ATP1A1 and ATP2B3 mutations compared with those without these or KCNJ5 mutations. Overexpression of Na+/K+-ATPase p.Gly99Arg and GIRK4 p.Trp126Arg in HAC15 adrenal cells resulted in upregulation of CYP11B2 gene expression and its transcriptional regulator NR4A2. Structural modeling of the Na+/K+-ATPase showed that the Gly99Arg substitution most likely interferes with the gateway to the ion binding pocket. In vitro functional assays demonstrated that Gly99Arg displays severely impaired ATPase activity, a reduced apparent affinity for Na+ activation of phosphorylation and K+ inhibition of phosphorylation that indicate decreased Na+ and K+ binding, respectively. Moreover, whole cell patch-clamp studies established that overexpression of Na+/K+-ATPase Gly99Arg causes membrane voltage depolarization. In conclusion, somatic mutations are common in APAs that result in an increase in CYP11B2 gene expression and may account for the dysregulated aldosterone production in a subset of patients with sporadic primary aldosteronism.