Fabrizio Buffolo

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.

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 (&rgr; = 0.70, P < 0.001 and &rgr; = 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.

Familial Hyperaldosteronism Type III

Primary aldosteronism is the most common form of endocrine hypertension. This disorder comprises both sporadic and familial forms. Four familial forms of primary aldosteronism (FH-I to FH-IV) have been described. FH-III is caused by germline mutations in KCNJ5, encoding the potassium channel Kir3.4 (also called GIRK4). These mutations alter the selectivity filter of the channel and lead to abnormal ion currents with loss of potassium selectivity, sodium influx and consequent increased intracellular calcium that causes excessive aldosterone biosynthesis. To date, eleven families have been reported, carrying six different mutations. Although the clinical features are variable, FH-III patients often display severe hyperaldosteronism with an early onset, associated with hypokalemia and diabetes insipidus-like symptoms. In most cases FH-III patients are resistant to pharmacological therapy and require bilateral adrenalectomy to control symptoms. In the present manuscript, we review the genetics and pathological basis of FH-III, the diagnostic work-up, clinical features and therapeutic management. Finally, we will describe a new case of FH-III of an Italian patient carrying a Gly151Arg mutation.

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.

GENETICS IN ENDOCRINOLOGY: The expanding genetic horizon of primary aldosteronism

Aldosterone is the main mineralocorticoid hormone in humans and plays a key role in maintaining water and electrolyte homeostasis. Primary aldosteronism (PA), characterized by autonomous aldosterone overproduction by the adrenal glands, affects 6% of the general hypertensive population and can be either sporadic or familial. Aldosterone-producing adenoma (APA) and bilateral adrenal hyperplasia (BAH) are the two most frequent subtypes of sporadic PA and 4 forms of familial hyperaldosteronism (FH-I to FH-IV) have been identified. Over the last six years, the introduction of next-generation sequencing has significantly improved our understanding of the molecular mechanisms responsible for autonomous aldosterone overproduction in both sporadic and familial PA. Somatic mutations in four genes (KCNJ5, ATP1A1, ATP2B3 and CACNA1D), differently implicated in intracellular ion homeostasis, have been identified in nearly 60% of the sporadic APAs. Germline mutations in KCNJ5 and CACNA1H cause FH-III and FH-IV, respectively, while germline mutations in CACNA1D cause the rare PASNA syndrome, featuring primary aldosteronism seizures and neurological abnormalities. Further studies are warranted to identify the molecular mechanisms underlying BAH and FH-II, the most common forms of sporadic and familial PA whose molecular basis is yet to be uncovered.

9B.06: COMPARISON BETWEEN ALDOSTERONE AND RENIN MEASUREMENT BY CHEMILUMINESCENT IMMUNOASSAY AND RADIOIMMUNOASSAY FOR THE DIAGNOSIS OF PRIMARY ALDOSTERONISM.

Objective: Primary aldosteronism (PA) is the most frequent cause of secondary hypertension responsible for an increased rate of cardiovascular events. According to the Endocrine Society Guidelines, up to 50% of hypertensive patients should be screened for PA, using the aldosterone to renin (or plasma renin activity, PRA) ratio (AARR and ARR, respectively). The automated Diasorin LIAISON® chemiluminescent immunoassay for renin and aldosterone measurement became available and in many laboratories is currently used instead of the classical radioimmunometric PRA and aldosterone assay. Aim of the study was to prospectively compare the diagnostic accuracy of AARR and ARR as screening test for PA and the two aldosterone assays also during confirmatory test in patients with a positive screening test. Design and method: One hundred patients were screened for PA and 44 patients underwent confirmatory test (either by intravenous saline load or by captopril challenge test). We considered as cut off for the AARR 2.7 (ng/dL/mU/L) and for the ARR 30 (ng/dL/ng/mL/h). All patients positive to one of the two screening test underwent confirmatory test; patients with positive confirmatory test underwent subtype diagnosis by CT scanning and adrenal vein sampling. Results: Seventy three patients were diagnosed as essential hypertensives, 22 had bilateral adrenal hyperplasia and 5 had an aldosterone producing adenomas (APA). The AARR displayed a sensitivity of 78% and a specificity of 100%, whereas the ARR had a sensitivity of 96% and a specificity of 90%. Of the 6/27 PA patients missed by AARR, none resulted to be affected by APA. All PA patients were correctly diagnosed by chemiluminescence at confirmatory test. In the overall sample of 181 measurements available both the correlation for the PRA with renin and for aldosterone in chemiluminescence and radioimmunoassay were highly significant (Rho = 0.66, p < 0.0001 and Rho = 0.80, p < 0.0001, respectively). On ROC curves, the AUC for AARR was 0.905 (95% CI 0.821-0.988) and for ARR 0.947 (95% CI 0.903–0.991) and they were not significantly different. Conclusions: The automated aldosterone and renin chemiluminescent assay is a reliable alternative to the well-established radioimmunometric method, especially for the detection of APA.

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.

Hyperaldosteronism: How to Discriminate Among Different Disease Forms?

Primary aldosteronism (PA), characterized by the inappropriate and abnormal adrenal secretion of aldosterone, is the most common cause of secondary hypertension. PA has been shown to increase cardiovasular and cerebrovascular risks in comparison with essential hypertension. PA is a multi-faceted disease, which comprises unilateral forms, benefitting from surgical treatment, and bilateral forms, which are the best managed medically. PA is more frequently sporadic, but in some cases, it displays a familial transmission pattern. For these reasons, it is important to diagnose PA early on and correctly distinguish and manage its different forms. In this review, we analyze the different forms of PA, with attention on the diagnostic pathway and the genetics of the disease.

Diagnostic approach to low-renin hypertension

Renin‐angiotensin‐aldosterone system (RAAS) plays a crucial role in maintaining water and electrolytes homoeostasis, and its deregulation contributes to the development of arterial hypertension. Since the historical description of the “classical” RAAS, a dramatic increase in our understanding of the molecular mechanisms underlying the development of both essential and secondary hypertension has occurred. Approximatively 25% of the patients affected by arterial hypertension display low‐renin levels, a definition that is largely arbitrary and depends on the investigated population and the specific characteristics of the assay. Most often, low‐renin levels are expression of a physiological response to sodium‐volume overload, but also a significant number of secondary hereditary or acquired conditions falls within this category. In a context of suppressed renin status, the concomitant examination of plasma aldosterone levels (which can be inappropriately elevated, within the normal range or suppressed) and plasma potassium are essential to formulate a differential diagnosis. To distinguish between the different forms of low‐renin hypertension is of fundamental importance to address the patient to the proper clinical management, as each subtype requires a specific and targeted therapy. The present review will discuss the differential diagnosis of the most common medical conditions manifesting with a clinical phenotype of low‐renin hypertension, enlightening the novelties in genetics of the familial forms.

A Case of Adrenal Vein Sampling in Primary Aldosteronism With Homolateral Suppression

Context: Adrenal venous sampling (AVS) is regarded as the gold standard for diagnosis of primary aldosteronism (PA) subtypes, although some authors have questioned its diagnostic accuracy and highlighted the lack of standardized procedure protocols and interpretation criteria for AVS. In particular, the usefulness of cosyntropin stimulation and benefit of superselective adrenal vein catheterization have been hotly debated. Objective: We report a case that highlights the potential pitfalls of superselective sampling and demonstrates a negligible effect of cosyntropin stimulation on aldosterone secretion in nonadenomatous adrenal tissue when an aldosterone-producing adenoma (APA) is present. Intervention and Results: A 38-year-old man with PA and a single right macroadenoma underwent AVS at our center. The procedure was performed both under basal conditions and during cosyntropin stimulation. Right adrenal vein angiography demonstrated two branches of the main adrenal vein trunk, one draining the nodule and one draining the right adrenal gland. Hormonal assays confirmed adrenal origin of left-sided and all right-sided samples, and were consistent with lateralization on the right side, with suppression of aldosterone secretion in the left adrenal gland and in the nonadenomatous right adrenal tissue. Cosyntropin-stimulated AVS results were similar to those of the unstimulated procedure. Conclusions: Cosyntropin stimulation does not significantly affect aldosterone secretion from nonadenomatous adrenal tissue when an APA is present and can therefore be used during AVS for PA. Superselective AVS should be performed with caution and interpreted by expert clinicians.