PhD Sheerazed Boulkroun
email: sheerazed.boulkroun@inserm.fr

Fabio Luiz Fernandes-Rosa

Maria-Christina Zennaro

Old and new genes in primary aldosteronism

Primary aldosteronism (PA) is the most common form of secondary hypertension affecting 5% to 10% of patients with arterial hypertension. In PA, high blood pressure is associated with high aldosterone and low renin levels, and often hypokalemia. In a majority of cases, autonomous aldosterone production by the adrenal gland is caused by an aldosterone producing adenoma (APA) or bilateral adrenal hyperplasia (BAH). During the last ten years, a better knowledge of the pathophysiology of PA came from the discovery of somatic and germline mutations in different genes in both sporadic and familial forms of the disease.

Those genes code for ion channel and pumps, as well as proteins involved in adrenal cortex development and function. Targeted next generation sequencing following immunohistochemistry guided detection of aldosterone synthase expression allows detection of somatic mutations in up to 90% of APA, while whole exome sequencing has discovered the genetic causes of four different familial forms of PA. The identification, in BAH, of somatic mutations in aldosterone producing cell clusters open new perspectives in our understanding of the bilateral form of the disease and the development of new therapeutic approaches.

Introduction

Aldosterone is synthesized by the zona glomerulosa of the adrenal cortex and plays a major role in regulating blood pressure, mainly by regulating sodium and water reabsorption in the kidney [START_REF] Shibata | YEARS OF THE MINERALOCORTICOID RECEPTOR: Mineralocorticoid receptor and NaCl transport mechanisms in the renal distal nephron[END_REF]. Effects of aldosterone on target tissues are mediated through its binding to the mineralocorticoid receptor (MR), a member of the nuclear receptor superfamily. The MR is a hormone dependent transcription factor, which binds to specific hormone responsive elements on regulatory regions of target genes. In the kidney, the MR is expressed in the aldosterone sensitive distal tubule, where aldosterone regulates sodium reabsorption and potassium excretion. In addition, MR is expressed also in other epithelial tissues such as the colon, sweat and salivary glands, where it regulates water and electrolyte homeostasis, and in nonepithelial tissues, such as the heart, vessels, and macrophages, where it mediates major deleterious effects of aldosterone excess on the cardiovascular system [START_REF] Jaisser | Emerging Roles of the Mineralocorticoid Receptor in Pathology: Toward New Paradigms in Clinical Pharmacology[END_REF].

Aldosterone biosynthesis

The adrenal cortex is composed of three distinct zones with different functions. The Zona Glomerulosa (ZG) forms the outer part of the cortex underneath the capsule, and produces mineralocorticoids, mainly aldosterone but also 11-deoxycorticosterone (DOC). Whereas aldosterone is produced only in ZG, due to specific expression of aldosterone synthase in this zone, DOC is produced in both ZG and ZF. The Zona Fasciculata (ZF), the thickest zone of the cortex, is involved in biosynthesis of the glucocorticoid hormone cortisol, which plays important roles in metabolism, stress response and inflammation. Finally, the Zona Reticularis (ZR), in contact with the medulla, produces sex steroids.

Aldosterone is synthetized from cholesterol, whose availability constitutes the limiting step in aldosterone biosynthesis. The translocation of cholesterol into the mitochondria, where it is converted into pregnenolone by the P450 side-chain cleavage enzyme (CYP11A1), is mediated by the steroidogenic acute regulatory protein (StAR). A cascade of enzymatic reactions then leads to the biosynthesis of aldosterone (Figure 1). Briefly, in ZG, the 3βhydroxysteroid dehydrogenase converts pregnenolone into progesterone and the 21hydroxylase catalyzes the 21-hydroxylation of progesterone to form 11-deoxycorticosterone.

These two enzymes are expressed throughout the adrenal cortex. The peculiar functional zonation of the adrenal cortex is due to zone-specific expression of different steroidogenic enzymes. In particular, the ZG expresses aldosterone synthase (encoded by CYP11B2), but not 17α-hydroxylase (encoded by the CYP17A1 gene) converting pregnenolone and progesterone to 17-OH-pregnenolone and 17-OH-progesterone); the ZF in contrast expresses both 17α-hydroxylase and 11β-hydroxylase (encoded by CYP11B1) converting 11deoxycortisol to cortisol (Figure 1). Thus in the ZG, aldosterone synthase catalyzes the three final enzymatic reactions leading to aldosterone biosynthesis: the 11β-hydroxylation of 11deoxycorticosterone, the 18-hydroxylation of corticosterone and finally the 18 methyloxidation of 18-OH-corticosterone to produce aldosterone [START_REF] Connell | The new biology of aldosterone[END_REF].

Physiological regulation of aldosterone biosynthesis

Aldosterone biosynthesis is under the control of many factors, the two most important being angiotensin II (AngII) and extracellular potassium concentration (K + ). Its biosynthesis is tightly controlled in order to maintain electrolyte homeostasis by the kidney. In absence of stimulation, ZG cells are hyperpolarized with a resting potential around -80mV. This is due to a dominant K + conductance due to the presence of a large number of potassium channels [START_REF] Bandulik | TASK1 and TASK3 potassium channels: determinants of aldosterone secretion and adrenocortical zonation[END_REF] and the Na + /K + -ATPase that maintain the K + gradient concentration between the plasma and the cytosol. Stimulation of ZG cells by AngII or increased plasma K + concentration lead to cell membrane depolarization, opening of voltage-gated Ca 2+ channels and increase of the intracellular Ca 2+ concentration. In parallel, binding of AngII to the angiotensin II Type 1 receptor (AT1R) stimulates inositol triphosphate-dependent Ca 2+ release from the endoplasmic reticulum. Increase of intracellular Ca 2+ concentration leads to activation of a phosphorylation cascade involving calmodulin and calcium/calmodulin-dependent protein kinases (CaMKI and/or CaMKIV) and results in the activation of Nurr1 and NGF1B, two transcription factors that positively regulate the transcription of CYP11B2 [START_REF] Bassett | The regulation of aldosterone synthase expression[END_REF][START_REF] Szekeres | Mechanisms of angiotensin II-mediated regulation of aldosterone synthase expression in H295R human adrenocortical and rat adrenal glomerulosa cells[END_REF].

In addition to these two major regulators, a number of other factors are known to induce or inhibit aldosterone biosynthesis. Although ACTH is known to be important in glucocorticoid biosynthesis, this hormone also contributes to the regulation of aldosterone biosynthesis [START_REF] Hattangady | Acute and chronic regulation of aldosterone production[END_REF].

Whereas acutely ACTH stimulates aldosterone biosynthesis via cAMP and activation of PKA signaling, chronically it inhibits aldosterone biosynthesis. One potential explanation for this phenomenon is the conversion by ACTH of ZG cells into ZF cells [START_REF] Crivello | Induction of cultured bovine adrenocortical zona glomerulosa cell 17-hydroxylase activity by ACTH[END_REF][START_REF] Hornsby | Functional and morphological observations on rat adrenal zona glomerulosa cells in monolayer culture[END_REF]. Serotonin (5HT) produced by mast cells present in the adrenal cortex has also been shown to stimulate aldosterone biosynthesis through binding to 5HT4R receptors and activation of the PKA signaling pathway [START_REF] Lefebvre | Production and metabolism of serotonin (5-HT) by the human adrenal cortex: paracrine stimulation of aldosterone secretion by 5-HT[END_REF]. In vitro data have shown that other factors can positively (Interleukine 6 [START_REF] Judd | Possible function of IL-6 and TNF as intraadrenal factors in the regulation of adrenal steroid secretion[END_REF], Epidermial Growth Factor [START_REF] Kim | EGF-stimulated aldosterone secretion is mediated by tyrosine phosphorylation but not by phospholipase C in cultured porcine adrenal glomerulosa cells[END_REF]) or negatively (Transforming Growth Factor-beta 1 [START_REF] Gupta | Transforming growth factor-beta 1 inhibits aldosterone biosynthesis in cultured bovine zona glomerulosa cells[END_REF], dopamine [START_REF] Mckenna | Dopamine inhibits angiotensinstimulated aldosterone biosynthesis in bovine adrenal cells[END_REF]) regulate aldosterone biosynthesis (for review, see [START_REF] Lefebvre | Paracrine Regulation of Aldosterone Secretion in Physiological and Pathophysiological Conditions[END_REF].

Recently, the adipocyte derived factor leptin has been shown also to be able to stimulate aldosterone biosynthesis in both cell and mouse models [START_REF] Faulkner | Leptin and Aldosterone[END_REF].

Primary aldosteronism

Primary aldosteronism (PA) is the most common form of secondary hypertension. Its prevalence was estimated between 5% to 10% of patients with arterial hypertension [START_REF] Hannemann | Prevalence of primary aldosteronism in patient's cohorts and in population-based studies--a review of the current literature[END_REF][START_REF] Rossi | A prospective study of the prevalence of primary aldosteronism in 1,125 hypertensive patients[END_REF] and up to 20% of patients with resistant hypertension [START_REF] Calhoun | Resistant hypertension[END_REF]. PA is described as a group of disorders in which aldosterone is inappropriately high considering salt status, relatively autonomous from the renin-angiotensin-system and non-suppressible by salt loading [START_REF] Funder | The Management of Primary Aldosteronism: Case Detection, Diagnosis, and Treatment: An Endocrine Society Clinical Practice Guideline[END_REF].

High blood pressure is associated to high aldosterone level and suppressed renin and often hypokalemia. The two major causes of PA are aldosterone producing adenoma (APA), first described by Jerome Conn in 1954 [START_REF] Conn | Primary aldosteronism[END_REF][START_REF] Conn | Man's endocrine and metabolic responses to stressing circumstances: clinical implications[END_REF], and bilateral adrenal hyperplasia (BAH), accounting together for more than 95% of cases. In rare cases, unilateral adrenal hyperplasia and adrenal carcinoma may be responsible for PA.

Patients with PA are more likely to develop cardiovascular complications than patients with primary hypertension matched for sex, age and blood pressure [START_REF] Rossi | Primary aldosteronism: cardiovascular, renal and metabolic implications[END_REF]. They have an increased rate of cardiovascular events such as stroke, myocardial infarction and atrial fibrillation [START_REF] Milliez | Evidence for an increased rate of cardiovascular events in patients with primary aldosteronism[END_REF].

They are also more likely to present left ventricular hypertrophy, diastolic dysfunction, alterations in left ventricular filling, nonfatal myocardial infarction, coronary artery disease, heart failure and atrial fibrillation [START_REF] Rossi | LVH in primary aldosteronism[END_REF][START_REF] Rossi | Remodeling of the left ventricle in primary aldosteronism due to Conn's adenoma[END_REF][START_REF] Savard | Cardiovascular complications associated with primary aldosteronism: a controlled cross-sectional study[END_REF]. This is due to the well-known deleterious effects of aldosterone on the heart and blood vessels [START_REF] Jaisser | Emerging Roles of the Mineralocorticoid Receptor in Pathology: Toward New Paradigms in Clinical Pharmacology[END_REF]. Despite publication of guidelines for the diagnosis, subtype identification and management of PA in 2008, and their update in 2016 [START_REF] Funder | The Management of Primary Aldosteronism: Case Detection, Diagnosis, and Treatment: An Endocrine Society Clinical Practice Guideline[END_REF][START_REF] Funder | Case detection, diagnosis, and treatment of patients with primary aldosteronism: an endocrine society clinical practice guideline[END_REF], the condition is still underdiagnosed or diagnosed years after the discovery of hypertension. This delay has a negative impact on the therapeutic outcome of adrenalectomy in patients with APA.

Over the last 10 years our knowledge on the genetic basis of PA has improved spectacularly and different new Mendelian forms of PA have been described. In parallel, somatic mutations in different genes have been discovered in APA as well as other aldosterone producing structures of the adrenal gland.

Familial forms of primary aldosteronism

Although the majority of cases of PA are sporadic, approximately 5% are found in a familial context and transmitted as an autosomal dominant trait [START_REF] Mulatero | Prevalence and characteristics of familial hyperaldosteronism: the PATOGEN study (Primary Aldosteronism in TOrino-GENetic forms)[END_REF]. Four different Mendelian forms of PA have been identified: Familial Hyperaldosteronism (FH) Type I to Type IV (Figure 2). However, while somatic mutations were identified in about 90% of APA, the genetic causes of an important number of familial forms are still unknown.

Familial Hyperaldosteronism Type I (FH-I)

In 1966, Sutherland and colleagues reported the case of a father and his son with moderate hypertension, hypokaelemia, increased aldosteronemia and suppressed plasma renin activity suggesting a Conn's syndrome [START_REF] Sutherland | Hypertension, increased aldosterone secretion and low plasma renin activity relieved by dexamethasone[END_REF]. Remarkably, dexamethasone treatment normalized their blood pressure, aldosterone and potassium levels and plasma renin activity [START_REF] Sutherland | Hypertension, increased aldosterone secretion and low plasma renin activity relieved by dexamethasone[END_REF]; more surprisingly, aldosterone biosynthesis was found to be sensitive to ACTH stimulation [START_REF] Ulick | Defective fasciculata zone function as the mechanism of glucocorticoid-remediable aldosteronism[END_REF].

These patients presented also high levels of 18-hydroxycortisol and 18-oxocortisol, their secretion being again under the control of ACTH and suppressible by glucocorticoids. FH-I is more often associated with bilateral adrenal hyperplasia rather than adrenocortical adenoma [START_REF] Stowasser | Familial hyperaldosteronism[END_REF][START_REF] Pascoe | Glucocorticoid-suppressible hyperaldosteronism and adrenal tumors occurring in a single French pedigree[END_REF].

In 1992, the molecular basis of this autosomal dominant form of PA was elucidated by Lifton and coll [START_REF] Lifton | A chimaeric 11 betahydroxylase/aldosterone synthase gene causes glucocorticoid-remediable aldosteronism and human hypertension[END_REF][START_REF] Lifton | Hereditary hypertension caused by chimaeric gene duplications and ectopic expression of aldosterone synthase[END_REF]. FH-I is due to unequal crossing-over between CYP11B1 and CYP11B2, coding for 11β-hydroxylase and aldosterone synthase, both localized on tandem in the same region of chromosome 8 and sharing 95% of sequence homology. This crossing-over leads to the fusion of the regulatory sequences of CYP11B1 to the coding sequence of CYP11B2, resulting in ectopic expression of aldosterone synthase in the ZF and regulation of its expression by ACTH (Figure 2A).

The prevalence of FH-I is estimated between 0.5% and 1% of cases of PA diagnosed in adult patients [START_REF] Mulatero | Prevalence and characteristics of familial hyperaldosteronism: the PATOGEN study (Primary Aldosteronism in TOrino-GENetic forms)[END_REF]; however the screening of a hypertensive pediatric population revealed that the prevalence of FH-I could represent 3% of cases [START_REF] Aglony | Frequency of familial hyperaldosteronism type 1 in a hypertensive pediatric population: clinical and biochemical presentation[END_REF].

Familial Hyperaldosteronism Type II (FH-II)

For many years FH-II was referring to familial forms of PA with an autosomal dominant mode of inheritance and for which FH-I had been excluded [START_REF] Gordon | Clinical and pathological diversity of primary aldosteronism, including a new familial variety[END_REF][START_REF] Stowasser | Primary aldosteronism: learning from the study of familial varieties[END_REF]. FH-II is clinically and biochemically indistinguishable from sporadic forms of PA and within the same family APA and bilateral adrenal hyperplasia may be present with variable phenotypes [START_REF] Mulatero | Prevalence and characteristics of familial hyperaldosteronism: the PATOGEN study (Primary Aldosteronism in TOrino-GENetic forms)[END_REF][START_REF] Stowasser | Familial varieties of primary aldosteronism[END_REF]. Diagnosis of FH-II is done on the basis of at least two affected members in the same family [START_REF] Medeau | Familial aspect of primary hyperaldosteronism: analysis of families compatible with primary hyperaldosteronism type 2[END_REF]. In some families a linkage was established with the chromosomal region 7p22 [START_REF] Lafferty | A novel genetic locus for low renin hypertension: familial hyperaldosteronism type II maps to chromosome 7 (7p22)[END_REF], however no mutations were found in different candidate genes located in this region [START_REF] Medeau | Familial aspect of primary hyperaldosteronism: analysis of families compatible with primary hyperaldosteronism type 2[END_REF][START_REF] Jeske | Examination of chromosome 7p22 candidate genes RBaK, PMS2 and GNA12 in familial hyperaldosteronism type II[END_REF]. The sequencing of other candidate genes including CYP11B2, AGTR1 coding for the AngII Receptor Type 1 and p53, a tumor-suppressor gene, was also negative [START_REF] Stowasser | Familial hyperaldosteronism[END_REF][START_REF] Ballantine | PCR-SSCP analysis of the p53 gene in tumours of the adrenal gland[END_REF][START_REF] Davies | Somatic mutations of the angiotensin II (AT1) receptor gene are not present in aldosterone-producing adenoma[END_REF].

Recently, whole exome sequencing performed in a large family first described by Michael Stowasser and colleagues [START_REF] Stowasser | Familial hyperaldosteronism type II: five families with a new variety of primary aldosteronism[END_REF] allowed the identification of a causal germline mutation in the CLCN2 gene (p.Arg172Gln). The same mutation was also found in three other families and four additional mutations (p.Tyr26Asn, p.Lys362del, p.Met22Lys, p.Ser865Arg) were identified in unrelated patients [START_REF] Scholl | CLCN2 chloride channel mutations in familial hyperaldosteronism type II[END_REF]. At the same time, Fernandes-Rosa and colleagues reported the identification of a de novo p.Gly24Asp mutation in CLCN2 in a 9-year-old girl presenting severe hypertension, profound hypokalemia, elevated plasma aldosterone and suppressed renin activity [START_REF] Fernandes-Rosa | A gain-offunction mutation in the CLCN2 chloride channel gene causes primary aldosteronism[END_REF] (Figure 2B).

CLCN2 encodes the ubiquitously expressed inwardly rectifying chloride channel CLC-2, that opens upon hyperpolarization, hypoosmotic cell swelling and acidic extracellular pH [START_REF] Thiemann | A chloride channel widely expressed in epithelial and non-epithelial cells[END_REF]. In humans, loss of function mutations in CLCN2 are responsible for leukodystrophy [START_REF] Depienne | Brain white matter oedema due to ClC-2 chloride channel deficiency: an observational analytical study[END_REF] that may also be associated with azoospermia [START_REF] Bella | Subclinical leukodystrophy and infertility in a man with a novel homozygous CLCN2 mutation[END_REF]. Similarly in mice, invalidation of Clcn2 leads to testicular degeneration, leukodystrophy as well as retinal degeneration [START_REF] Blanz | Leukoencephalopathy upon disruption of the chloride channel ClC-2[END_REF][START_REF] Bosl | Male germ cells and photoreceptors, both dependent on close cell-cell interactions, degenerate upon ClC-2 Cl(-) channel disruption[END_REF].

Interestingly, mutations identified in PA result in a gain of function of the channel, leading to increased chloride currents. In particular, the p.Gly24Asp mutation is located in a wellconserved inactivation domain of the protein and abolishes the voltage-and time-dependent gating of the channel, strongly increasing chloride conductance at resting potentials [START_REF] Fernandes-Rosa | A gain-offunction mutation in the CLCN2 chloride channel gene causes primary aldosteronism[END_REF].

Increased efflux of Cl -ions leads to cell membrane depolarization, opening of voltage gated Ca 2+ channel and ultimately increased aldosterone biosynthesis [START_REF] Scholl | CLCN2 chloride channel mutations in familial hyperaldosteronism type II[END_REF][START_REF] Fernandes-Rosa | A gain-offunction mutation in the CLCN2 chloride channel gene causes primary aldosteronism[END_REF]. Different mutations have slightly different effects on channel function. In particular, the p.Arg172Gln mutation seems to be milder than other mutations when tested in two-electrode voltage clamp studies in Xenopus oocytes [START_REF] Goppner | Pathogenesis of hypertension in a mouse model for human CLCN2 related hyperaldosteronism[END_REF], explaining the incomplete penetrance and phenotypic heterogeneity observed in affected family members [START_REF] Scholl | CLCN2 chloride channel mutations in familial hyperaldosteronism type II[END_REF]. Similar to the human disease, mice expressing a constitutively open CLC-2 chloride channel, recapitulating gain of function mutations found in FH-II, display marked hypertension, high aldosterone levels, low renin activity and hypokalemia, the principal features of human PA [START_REF] Goppner | Pathogenesis of hypertension in a mouse model for human CLCN2 related hyperaldosteronism[END_REF]. This is due to increased chloride conductance of zona glomerulosa cells, leading to a strong cell membrane depolarization and increased cytoplasmic calcium concentration, followed by increased expression of aldosterone synthase, thus confirming the role of CLC2 mutations in the pathogenesis of PA. A similar phenotype is observed in mice carrying a mutation at the position homologous to p.Arg172Gln. Heterozygous Clcn2 R180Q/+ mice show increased aldosterone levels and Cyp11b2 expression; in addition, male mice have increased aldosterone to renin ratio and slightly increase blood pressure [START_REF] Schewe | Elevated aldosterone and blood pressure in a mouse model of familial hyperaldosteronism with ClC-2 mutation[END_REF] In contrast, Clcn2 knockout mice show normal aldosterone levels and Cyp11b2 expression, but increased plasma renin concentration, suggesting that activation of the renin angiotensin system maintains normal aldosterone levels in these animals and that CLC-2 plays a role in regulating physiological aldosterone biosynthesis [START_REF] Schewe | Elevated aldosterone and blood pressure in a mouse model of familial hyperaldosteronism with ClC-2 mutation[END_REF].

Familial Hyperaldosteronism Type III (FH-III)

In 2008, Geller and coll reported a new familial form of primary aldosteronism in a father and his two daughters [START_REF] Geller | A novel form of human mendelian hypertension featuring nonglucocorticoid-remediable aldosteronism[END_REF]. At 5 years of age, the father presented severe hypertension, high aldosterone, profound hypokalemia and evidence of left ventricular hypertrophy associated with adrenal hyperplasia [START_REF] Therien | Primary aldosteronism due to adrenal hyperplasia; occurrence in a boy aged 10 years[END_REF]. He underwent bilateral adrenalectomy four years later. His two daughters presented at 7 and 4 years of age severe hypertension associated with profound hypokalemia, elevated plasma aldosterone despite suppressed renin activity. Only the older girl presented also concentric left ventricular hypertrophy with preserved ejection fraction.

These three patients presented also high levels of the hybrid steroids 18-oxocortisol and 18hydroxycortisol. However, lack of suppression of aldosterone by dexamethasone excluded FH-I. Despite multiple therapies, both patients had to undergo bilateral adrenalectomy to achieve normalization of BP and kalemia. Their adrenals presented ZF hyperplasia and atrophy of the ZG, with CYP11B2 expression throughout the adrenal cortex and coexpression of CYP11B2, CYP11B1 and CYP17A1, suggesting that aldosterone and hybrid steroid production was due to ectopic expression of aldosterone synthase in the ZF [START_REF] Geller | A novel form of human mendelian hypertension featuring nonglucocorticoid-remediable aldosteronism[END_REF][START_REF] Gomez-Sanchez | Disordered zonal and cellular CYP11B2 enzyme expression in familial hyperaldosteronism type 3[END_REF].

The molecular abnormality responsible for this new form of familial PA is the mutation p.Thr158Ala in the KCNJ5 gene, encoding the G protein-activated inward rectifier potassium channel GIRK4 [START_REF] Choi | K+ channel mutations in adrenal aldosterone-producing adenomas and hereditary hypertension[END_REF] (Figure 2C). The mutation is located in the selectivity filter of GIRK4 and modifies its ion selectivity, with the loss of K + selectivity in favor of sodium and increase in Na + influx into the cell. Different germline mutations in KCNJ5 were identified in familial cases of PA, with a correlation between the type of mutation and the severity of the disease.

Whereas a severe phenotype with early-onset hypertension resistant to treatment was associated with the mutations p.Gly151Arg, p.Thr158Ala and p.Ile157Ser [START_REF] Choi | K+ channel mutations in adrenal aldosterone-producing adenomas and hereditary hypertension[END_REF][START_REF] Scholl | Hypertension with or without adrenal hyperplasia due to different inherited mutations in the potassium channel KCNJ5[END_REF][START_REF] Charmandari | A Novel Point Mutation in the KCNJ5 Gene Causing Primary Hyperaldosteronism and Early-Onset Autosomal Dominant Hypertension[END_REF], a milder phenotype was observed in patients carrying the mutations p.Gly151Glu [START_REF] Scholl | Hypertension with or without adrenal hyperplasia due to different inherited mutations in the potassium channel KCNJ5[END_REF][START_REF] Mulatero | KCNJ5 mutations in European families with nonglucocorticoid remediable familial hyperaldosteronism[END_REF] and p.Tyr152Cys [START_REF] Monticone | a Novel Y152C KCNJ5 mutation responsible for familial hyperaldosteronism type III[END_REF]. These observations were also supported by in vitro data showing that the p.Gly151Glu affects the GIRK4 channel selectivity similarly to other mutations; however the resulting Na + conductance was larger for this mutation than for the others, leading to rapid cell lethality probably limiting cell proliferation [START_REF] Scholl | Hypertension with or without adrenal hyperplasia due to different inherited mutations in the potassium channel KCNJ5[END_REF]. However, this phenotype-genotype association between KCNJ5 mutations and the severity of the disease was questioned by the description of a sporadic patient with FH-III harboring the p.Gly151Arg mutation that was associated to a severe phenotype. Against all odds, this patient responded well to spironolactone and no adrenal enlargement was observed after seven years of follow-up [START_REF] Adachi | Discordant genotype-phenotype correlation in familial hyperaldosteronism type III with KCNJ5 gene mutation: a patient report and review of the literature[END_REF].

FH-III is a rare disease, its prevalence is estimated to be less than 0.5% of cases of PA diagnosed in adult patients and around 8% among familial forms [START_REF] Mulatero | KCNJ5 mutations in European families with nonglucocorticoid remediable familial hyperaldosteronism[END_REF][START_REF] Monticone | GENETICS IN ENDOCRINOLOGY: The expanding genetic horizon of primary aldosteronism[END_REF][START_REF] Monticone | Familial hyperaldosteronism type III[END_REF].

Familial Hyperaldosteronism Type IV (FH-IV)

Exome sequencing performed on 40 unrelated patients with early onset hypertension due to PA before age 10 years allowed the identification of a germline mutation in the voltage gated T-type calcium channel Cav3.2 (p.Met1549Val), encoded by CACNA1H, in five patients [START_REF] Scholl | Recurrent gain of function mutation in calcium channel CACNA1H causes early-onset hypertension with primary aldosteronism[END_REF].

A similar approach led to the identification of four different mutations (p.Met1549Ile, p.Ser196Leu, p.Pro2083Leu and p.Val1951Glu) in the same gene in patients with early onset and familial PA [START_REF] Daniil | CACNA1H Mutations Are Associated With Different Forms of Primary Aldosteronism[END_REF] (Figure 2D). These mutations are inherited in an autosomal dominant manner with incomplete penetrance of the disease, including asymptomatic patients. Different CACNA1H variants have been associated with epileptetic disorders [START_REF] Chen | Association between genetic variation of CACNA1H and childhood absence epilepsy[END_REF][START_REF] Heron | Extended spectrum of idiopathic generalized epilepsies associated with CACNA1H functional variants[END_REF][START_REF] Khosravani | Gating effects of mutations in the Cav3.2 T-type calcium channel associated with childhood absence epilepsy[END_REF][START_REF] Khosravani | Effects of Cav3.2 channel mutations linked to idiopathic generalized epilepsy[END_REF], autism spectrum disorders [START_REF] Splawski | CACNA1H mutations in autism spectrum disorders[END_REF] and chronic pain [START_REF] Souza | Two heterozygous Cav3.2 channel mutations in a pediatric chronic pain patient: recording condition-dependent biophysical effects[END_REF]. Accordingly, one patient carrying a p.Met1549Ile mutation was diagnosed with multiplex developmental disorder at age 10 years [START_REF] Daniil | CACNA1H Mutations Are Associated With Different Forms of Primary Aldosteronism[END_REF] and two patients carrying the p.Met1549Val mutation presented developmental delay or attention deficit [START_REF] Scholl | Recurrent gain of function mutation in calcium channel CACNA1H causes early-onset hypertension with primary aldosteronism[END_REF]. Phenotypic variability was observed among patients carrying different CACNA1H mutation, some patients being diagnosed with FH-II and some others with sporadic PA or APA. This was also observed among member of the same family carrying the same CACNA1H mutation and was attributed to age-dependent variability or to genetic or environmental modifiers [START_REF] Scholl | Recurrent gain of function mutation in calcium channel CACNA1H causes early-onset hypertension with primary aldosteronism[END_REF]. All the mutations identified in Cav3.2 affect significantly the electrophysiological properties of the channel, modifying their activation and/or inactivation properties, their steady-state inactivation and/or their voltage-dependent facilitation [START_REF] Scholl | Recurrent gain of function mutation in calcium channel CACNA1H causes early-onset hypertension with primary aldosteronism[END_REF][START_REF] Daniil | CACNA1H Mutations Are Associated With Different Forms of Primary Aldosteronism[END_REF].

These functional modifications lead to shift of the voltage dependence of the channel towards more negative potentials, increasing calcium influx into the cell, with consequent activation of calcium signaling and aldosterone production [START_REF] Scholl | Recurrent gain of function mutation in calcium channel CACNA1H causes early-onset hypertension with primary aldosteronism[END_REF][START_REF] Daniil | CACNA1H Mutations Are Associated With Different Forms of Primary Aldosteronism[END_REF][START_REF] Reimer | CACNA1H(M1549V) Mutant Calcium Channel Causes Autonomous Aldosterone Production in HAC15 Cells and Is Inhibited by Mibefradil[END_REF].

Other genetic forms of Hyperaldosteronism

De novo germline mutations in CACNA1D (p.Gly403Asp and p.Ile770Met), encoding the α1 subunit of the L-type voltage gated calcium channel Cav1.3, were identified in two children with early-onset PA with no adrenal abnormality [START_REF] Scholl | Somatic and germline CACNA1D calcium channel mutations in aldosterone-producing adenomas and primary aldosteronism[END_REF]. These mutations induce a gain of function, allowing the channel to open at lower voltages and leading to increased intracellular calcium concentration. In these two young patients, PA was associated with seizures and neurologic abnormalities (PASNA for Primary Aldosteronism associated with Seizures and Neurologic Abnormalities).

Genetic testing in Familial Hyperaldosteronism

Genetic testing for Familial Hyperaldosteronism has been proposed in different categories of patients [START_REF] Funder | The Management of Primary Aldosteronism: Case Detection, Diagnosis, and Treatment: An Endocrine Society Clinical Practice Guideline[END_REF][START_REF] Amar | SFE/SFHTA/AFCE primary aldosteronism consensus: Introduction and handbook[END_REF][START_REF] Zennaro | SFE/SFHTA/AFCE consensus on primary aldosteronism, part 5: Genetic diagnosis of primary aldosteronism[END_REF]. Genetic testing for FH-I is recommended in patients with onset of PA before 20 years of age and/or a family history of PA or strokes before 40 years of age.

Familial screening should also be performed in relatives of patients with confirmed FH-I, as adverse cardiovascular damage by aldosterone may precede the onset of high blood pressure [START_REF] Amar | SFE/SFHTA/AFCE primary aldosteronism consensus: Introduction and handbook[END_REF][START_REF] Stowasser | Evidence for abnormal left ventricular structure and function in normotensive individuals with familial hyperaldosteronism type I[END_REF]. Genetic screening for FH-III is recommended in young patients with PA [START_REF] Funder | The Management of Primary Aldosteronism: Case Detection, Diagnosis, and Treatment: An Endocrine Society Clinical Practice Guideline[END_REF][START_REF] Amar | SFE/SFHTA/AFCE primary aldosteronism consensus: Introduction and handbook[END_REF][START_REF] Zennaro | SFE/SFHTA/AFCE consensus on primary aldosteronism, part 5: Genetic diagnosis of primary aldosteronism[END_REF]. Genetic screening for FH-IV may be performed in patients with early onset hypertension and PA before the age of 10 years, while mutations in CACNA1D have to be searched for in patients with early onset hypertension and PA associated with complex neurologic syndrome with seizures [START_REF] Funder | The Management of Primary Aldosteronism: Case Detection, Diagnosis, and Treatment: An Endocrine Society Clinical Practice Guideline[END_REF][START_REF] Amar | SFE/SFHTA/AFCE primary aldosteronism consensus: Introduction and handbook[END_REF][START_REF] Zennaro | SFE/SFHTA/AFCE consensus on primary aldosteronism, part 5: Genetic diagnosis of primary aldosteronism[END_REF]. The recent discovery of CLCN2 mutations in FH-II strongly supports the addition of this gene among those tested, if the criteria for a genetic form are strong and other inherited forms were excluded. Practically speaking, it is difficult to clearly separate the different familial forms of PA based on clinical and biochemical data, as there is important clinical heterogeneity and overlap between them. Therefore, the main criteria for performing genetic testing are a young age of onset and/or a family history of PA. Genetic screening may allow identifying patients for targeted treatment and exclude patients from undergoing AVS and possibly unilateral adrenalectomy in case of asymmetric aldosterone production. It also allows better management of affected families and prevention of cardiovascular complications.

Somatic mutations in Aldosterone Producing Adenoma

Whole exome sequencing studies performed on DNA from APA and paired germline DNA led to the identification of recurrent somatic mutations in genes coding for ion channels (KCNJ5 and CACNA1D) and ATPases (ATP1A1 and ATP2B3) in a majority of APA [START_REF] Choi | K+ channel mutations in adrenal aldosterone-producing adenomas and hereditary hypertension[END_REF][START_REF] Scholl | Somatic and germline CACNA1D calcium channel mutations in aldosterone-producing adenomas and primary aldosteronism[END_REF][START_REF] Azizan | Somatic mutations in ATP1A1 and CACNA1D underlie a common subtype of adrenal hypertension[END_REF][START_REF] Beuschlein | Somatic mutations in ATP1A1 and ATP2B3 lead to aldosterone-producing adenomas and secondary hypertension[END_REF]. Similarly to familial forms, these mutations increase aldosterone biosynthesis by affecting cell membrane potential and/or intracellular ion homeostasis in zona glomerulosa cells. This ultimately increases intracellular calcium concentration and activates calcium signaling, increasing CYP11B2 expression and aldosterone production.

In 2011, Choi and co-workers performed whole exome sequencing in 22 APA, identifying two different somatic mutations (p.Gly151Arg and p.Leu168Arg) affecting the KCNJ5 gene in 8 APA [START_REF] Choi | K+ channel mutations in adrenal aldosterone-producing adenomas and hereditary hypertension[END_REF]. Using a similar strategy, somatic mutations were identified in ATP1A1, coding for the α subunit of the Na + ,K + -ATPase [START_REF] Azizan | Somatic mutations in ATP1A1 and CACNA1D underlie a common subtype of adrenal hypertension[END_REF][START_REF] Beuschlein | Somatic mutations in ATP1A1 and ATP2B3 lead to aldosterone-producing adenomas and secondary hypertension[END_REF], ATP2B3 coding for the plasma membrane calcium ATPase 3 [START_REF] Beuschlein | Somatic mutations in ATP1A1 and ATP2B3 lead to aldosterone-producing adenomas and secondary hypertension[END_REF] and CACNA1D, coding for the voltage gated calcium channel Cav1.3 [START_REF] Scholl | Somatic and germline CACNA1D calcium channel mutations in aldosterone-producing adenomas and primary aldosteronism[END_REF][START_REF] Azizan | Somatic mutations in ATP1A1 and CACNA1D underlie a common subtype of adrenal hypertension[END_REF]. The KCNJ5 mutations affect GIRK4 ion selectivity and lead to sodium influx into the cell. ATP1A1 mutations lead to loss of pump activity and inward proton and sodium leak and intracellular acidification [START_REF] Stindl | Pathogenesis of Adrenal Aldosterone-Producing Adenomas Carrying Mutations of the Na(+)/K(+)-ATPase[END_REF]. The resulting chronic cell membrane depolarization induces opening of voltage-dependant calcium channels with activation of calcium signalling and stimulation of aldosterone production. Mutations in ATP2B3 lead to a reduced calcium export from the cell as well as increased calcium influx due to different mechanisms [START_REF] Tauber | Cellular Pathophysiology of an Adrenal Adenoma-Associated Mutant of the Plasma Membrane Ca(2+)-ATPase ATP2B3[END_REF],

while mutations in CACNA1D lead to channel opening at less depolarized membrane potentials. Again, these mutations increase intracellular calcium signalling.

The prevalence of somatic mutations in APA was investigated in different studies by Sanger sequencing on DNA extracted from nodules identified macroscopically [START_REF] Boulkroun | Prevalence, Clinical, and Molecular Correlates of KCNJ5 Mutations in Primary Aldosteronism[END_REF][START_REF] Fernandes-Rosa | Genetic spectrum and clinical correlates of somatic mutations in aldosteroneproducing adenoma[END_REF][START_REF] Scholl | Novel somatic mutations in primary hyperaldosteronism are related to the clinical, radiological and pathological phenotype[END_REF][START_REF] Akerstrom | Novel somatic mutations and distinct molecular signature in aldosterone-producing adenomas[END_REF][START_REF] Wang | Prevalence and characterization of somatic mutations in Chinese aldosterone-producing adenoma patients[END_REF][START_REF] Mohideen | Prevalence and Histopathological Characteristics of KCNJ5 Mutant Aldosterone-Producing Adenomas in a Multi-Ethnic Malaysian Cohort[END_REF]. The largest study, performing target sequencing of hot spot regions of APA driver genes in 474 APA, identified somatic mutations in 54% of samples [START_REF] Fernandes-Rosa | Genetic spectrum and clinical correlates of somatic mutations in aldosteroneproducing adenoma[END_REF]. KCNJ5 mutations were found in 38% of APA, CACNA1D in 9.3%, ATP1A1 in 5.3%, and ATP2B3 in 1.7% of the samples (Table 1).

Recently, a different sequencing approach using full-gene-based next generation sequencing (NGS) performed on DNA extracted from aldosterone synthase positive regions on formalinfixed paraffin-embedded (FFPE) tissues completely changed the prevalence of somatic mutations in APA. This approach has allowed identifying somatic mutations in up to 88% of APA, showing that in some cases APA were satellite small nodules neighboring large adenomas without CYP11B2 expression [START_REF] Nanba | Targeted Molecular Characterization of Aldosterone-Producing Adenomas in White Americans[END_REF][START_REF] Nanba | Genetic Characteristics of Aldosterone-Producing Adenomas in Blacks[END_REF][START_REF] Sousa | Cellular and Molecular Heterogeneity in Adrenals with Aldosterone Producing Adenoma[END_REF] (Table 1).

In the majority of studies, somatic KCNJ5 mutations are the most prevalent, with an overall prevalence of 43% in a meta-analysis of somatic KCNJ5 mutations comprising 1636 patients with APA [START_REF] Lenzini | A Meta-Analysis of Somatic KCNJ5 K(+) Channel Mutations In 1636 Patients With an Aldosterone-Producing Adenoma[END_REF]. Ethnic differences were observed among cohorts. The frequency of somatic KCNJ5 mutations ranges from 40% in European and white American cohorts to up to 76% in Asian cohorts [START_REF] Wang | Prevalence and characterization of somatic mutations in Chinese aldosterone-producing adenoma patients[END_REF][START_REF] Taguchi | Expression and mutations of KCNJ5 mRNA in Japanese patients with aldosteroneproducing adenomas[END_REF][START_REF] Zheng | Clinical characteristics of somatic mutations in Chinese patients with aldosterone-producing adenoma[END_REF][START_REF] Wu | Prevalence and clinical correlates of somatic mutation in aldosterone producing adenoma-Taiwanese population[END_REF]. In the meta-analysis, the frequency of somatic KCNJ5 mutations was 63% in patients from East Asia and 35% in patients from Europe + the United States + Australia [START_REF] Lenzini | A Meta-Analysis of Somatic KCNJ5 K(+) Channel Mutations In 1636 Patients With an Aldosterone-Producing Adenoma[END_REF]. Moreover, somatic mutations in CACNA1D are most frequent in APA from African-American patients (42%) while the frequency of somatic KCNJ5 mutations in this population was 34% [START_REF] Nanba | Genetic Characteristics of Aldosterone-Producing Adenomas in Blacks[END_REF]. The CYP11B2 immunohistochemistry-guided next-generation sequencing (IHC-guided NGS) approach has allowed the identification of a larger number of somatic CACNA1D and ATP1A1 mutations in APA. In one cohort of white Americans, the prevalence of ATP1A1 and CACNA1D mutations was 21% and 17%, respectively [START_REF] Nanba | Targeted Molecular Characterization of Aldosterone-Producing Adenomas in White Americans[END_REF], much higher than in previous studies performing Sanger sequencing [START_REF] Fernandes-Rosa | Genetic spectrum and clinical correlates of somatic mutations in aldosteroneproducing adenoma[END_REF][START_REF] Scholl | Novel somatic mutations in primary hyperaldosteronism are related to the clinical, radiological and pathological phenotype[END_REF][START_REF] Akerstrom | Novel somatic mutations and distinct molecular signature in aldosterone-producing adenomas[END_REF]. The frequency of ATP2B3 mutations ranges from 1.7% to 4% in different cohorts [84-86, 89, 90]. In addition to these well characterized mutations, a small proportion of APA also carries mutations in CTNNB1, coding for β-catenin. The frequency of CTNNB1 mutations ranges from 2.1% to 5.1 % [START_REF] Scholl | Novel somatic mutations in primary hyperaldosteronism are related to the clinical, radiological and pathological phenotype[END_REF][START_REF] Nanba | Targeted Molecular Characterization of Aldosterone-Producing Adenomas in White Americans[END_REF][START_REF] Nanba | Genetic Characteristics of Aldosterone-Producing Adenomas in Blacks[END_REF][START_REF] Akerstrom | Activating mutations in CTNNB1 in aldosterone producing adenomas[END_REF][START_REF] Wu | The prevalence of CTNNB1 mutations in primary aldosteronism and consequences for clinical outcomes[END_REF], but may be higher in women with PA during pregnancy of after menopause [START_REF] Teo | Pregnancy, Primary Aldosteronism, and Adrenal CTNNB1 Mutations[END_REF]. Finally, a somatic CLCN2 mutation was recently described in one APA [START_REF] Dutta | Case report: A somatic mutation in CLCN2 identified in a sporadic aldosterone producing adenoma[END_REF], while somatic PRKACA (coding for the catalytic subunit of protein kinase A) mutations were observed in two patients, one with biochemical findings of aldosterone and cortisol cosecretion [START_REF] Rhayem | PRKACA Somatic Mutations Are Rare Findings in Aldosterone-Producing Adenomas[END_REF].

Several studies aimed at identifying clinical and biochemical surrogate markers of the somatic mutation status, allowing to predict mutations of APA before surgery. Analyzing the clinical correlates of KCNJ5 mutations in 1636 patients from 13 studies, Lenzini et al [START_REF] Lenzini | A Meta-Analysis of Somatic KCNJ5 K(+) Channel Mutations In 1636 Patients With an Aldosterone-Producing Adenoma[END_REF] showed that carriers of KCNJ5 mutations were younger and more often females, associations largely observed in other studies, including studies using CYP11B2 IHC-guided NGS [83-86, 89, 90, 101, 102]. KCNJ5 mutations were also associated with higher preoperative aldosterone levels and larger tumor size [START_REF] Lenzini | A Meta-Analysis of Somatic KCNJ5 K(+) Channel Mutations In 1636 Patients With an Aldosterone-Producing Adenoma[END_REF], although these correlations were not observed in all cohorts [START_REF] Fernandes-Rosa | Genetic spectrum and clinical correlates of somatic mutations in aldosteroneproducing adenoma[END_REF].

The mutation status may influence the diagnosis of PA, the therapeutic options and the outcome, including cardiovascular complications. Association of KCNJ5 mutations with higher lateralization index at adrenal venous sampling (AVS) was described, suggesting increased aldosterone production on the APA side and suppression on the contralateral adrenal [START_REF] Seccia | Somatic mutations in the KCNJ5 gene raise the lateralization index: implications for the diagnosis of primary aldosteronism by adrenal vein sampling[END_REF]. This association was not replicated in a subsequent study [START_REF] Osswald | Lack of influence of somatic mutations on steroid gradients during adrenal vein sampling in aldosterone-producing adenoma patients[END_REF]. Concerning the identification of somatic mutations before surgery and a potential impact for the selection of PA patients undergoing AVS, Williams et al described a steroid fingerprint in peripheral vein plasma able to correctly classify the somatic genotype of 92% of APA [START_REF] Williams | Genotype-Specific Steroid Profiles Associated With Aldosterone-Producing Adenomas[END_REF]. In addition, based on the PASO criteria for APA outcome [START_REF] Williams | Outcomes after adrenalectomy for unilateral primary aldosteronism: an international consensus on outcome measures and analysis of remission rates in an international cohort[END_REF], KCNJ5 mutations were shown to be an independent predictor of hypertension cure after adrenalectomy [START_REF] Vilela | KCNJ5 Somatic Mutation Is a Predictor of Hypertension Remission After Adrenalectomy for Unilateral Primary Aldosteronism[END_REF]. Finally, KCNJ5 mutations were correlated to more prominent cardiovascular damage and higher left ventricular mass in an Italian study [START_REF] Rossi | KCNJ5 gene somatic mutations affect cardiac remodelling but do not preclude cure of high blood pressure and regression of left ventricular hypertrophy in primary aldosteronism[END_REF], and significant postoperative improvements in left ventricular mass in Japanese subjects [START_REF] Kitamoto | Comparison of cardiovascular complications in patients with and without KCNJ5 gene mutations harboring aldosterone-producing adenomas[END_REF]. The discovery of somatic mutations in APA driver genes also opens new perspectives in PA care, particularly in therapeutic options targeting mutant ion channels. It has been shown that high therapeutic doses of calcium channel blockers such as verapamil are able to block a KCNJ5 mutant channel in vitro [START_REF] Tauber | Pharmacology and pathophysiology of mutated KCNJ5 found in adrenal aldosteroneproducing adenomas[END_REF].

In addition, recent studies revealed that macrolides may also inhibit mutant KCNJ5 channels, decreasing CYP11B2 expression and aldosterone production both in the adrenocortical HAC15 cell line [START_REF] Scholl | Macrolides selectively inhibit mutant KCNJ5 potassium channels that cause aldosteroneproducing adenoma[END_REF] and in primary culture of APA harboring KCNJ5 mutations [START_REF] Caroccia | Macrolides Blunt Aldosterone Biosynthesis: A Proof-of-Concept Study in KCNJ5 Mutated Adenoma Cells Ex Vivo[END_REF].

Currently, study of the potential use of macrolides for the preoperative identification of KCNJ5 positive APA and for PA subtype diagnosis is ongoing [START_REF] Maiolino | Macrolides for KCNJ5-mutated aldosterone-producing adenoma (MAPA): design of a study for personalized diagnosis of primary aldosteronism[END_REF].

Somatic mutations in other aldosterone producing structures in the adrenal gland

CYP11B2 expression is not only found in a limited number of ZG cells, but also in specific morphological structures with a defined boundary from the neighboring areas, but without an apparent fibrous capsule, called Aldosterone Producing Cell Clusters (APCC) [START_REF] Nishimoto | Adrenocortical zonation in humans under normal and pathological conditions[END_REF][START_REF] Boulkroun | Adrenal cortex remodeling and functional zona glomerulosa hyperplasia in primary aldosteronism[END_REF].

These specific structure express CYP11B2 and 3β-HSD2, but not Disabled-2, a marker of ZG cells, or CYP11B1, a marker of ZF cells, suggesting that APCC may constitute an intermediate structure between ZG and ZF cells [START_REF] Nishimoto | Adrenocortical zonation in humans under normal and pathological conditions[END_REF][START_REF] Boulkroun | Adrenal cortex remodeling and functional zona glomerulosa hyperplasia in primary aldosteronism[END_REF]. APCC were described in normal adrenal cortex as well as in adrenal cortex adjacent to an APA; they have been proposed to constitute renin-independent aldosterone producing structures.

RNA-sequencing revealed that APCC have a molecular profile very similar to the ZG, but different from ZF samples [START_REF] Nishimoto | Aldosterone-stimulating somatic gene mutations are common in normal adrenal glands[END_REF]. CYP11B2 immuhistochemistry guided NGS of FFPE adrenal tissue allowed to sequence APCC from normal adrenal glands and to identify somatic mutations in CACNA1D, ATP1A1 and ATP2B3 [START_REF] Nishimoto | Aldosterone-stimulating somatic gene mutations are common in normal adrenal glands[END_REF]. Based on this finding, it was hypothesised that APA derive from APCC. Interestingly, this hypothesis was reinforced by the identification of specific lesions called APCC-to-APA translational lesions (pAATL); these structures are composed of a sub-capsular APCC-like structure and an inner micro-APA-like (mAPA-like) structure [START_REF] Nishimoto | Immunohistochemistry of aldosterone synthase leads the way to the pathogenesis of primary aldosteronism[END_REF]. Mutations in APA driver genes were identified in different parts of pAATL; in one case a somatic KCNJ5 mutation was identified only in the mAPA-like structure, whereas in another case a somatic ATP1A1 mutation was identified in the entire structure [START_REF] Nishimoto | Immunohistochemistry of aldosterone synthase leads the way to the pathogenesis of primary aldosteronism[END_REF]. Taken together, these results suggest that APA with ATP1A1, ATP2B3 and CACNA1D mutations may derive from APCC, however the sequence of events leading to APA development remains unknown.

Recently, the presence of APCC was also described in a cohort of 15 patients with bilateral adrenal hyperplasia who underwent unilateral adrenalectomy [START_REF] Omata | Cellular and Genetic Causes of Idiopathic Hyperaldosteronism[END_REF]. All adrenals harbored at least one APCC; sequencing of 99 APCC from 14 out of 15 of these adrenals revealed the presence of somatic mutations in at least one APCC per adrenal. In contrast to the prevalence of mutations observed in APA, mutations are almost exclusively found in CACNA1D [START_REF] Omata | Cellular and Genetic Causes of Idiopathic Hyperaldosteronism[END_REF].

Interestingly, the number of APCC was significantly higher in adrenals with BAH than in adrenals from normotensive controls, suggesting that accumulation of APCC harboring somatic mutations in APA driver genes may contribute to BAH. Despite a small number of samples, and the need for replication in other cohorts of patients, this study for the first time identified a potential role for APCC in the development of BAH.

Conclusions

During the last ten years, whole exome sequencing allowed to decipher the genetic basis of familial and sporadic forms of PA. Until recently, the frequency of somatic mutations in APA was considered to be ≈50%. However, the recent application of CYP11B2immunohistochemistry guided NGS revealed the presence of somatic mutations in known APA driver genes in up to 90% of APA, suggesting that a majority of APA is due to mutations in known genes. Our understanding of inherited forms of PA has been significantly improved with the identification of germline mutations in KCNJ5, CACNA1H and CLCN2 and the definition of four familial forms of hyperaldosteronism, FH-I to FH-IV. Nevertheless, the genetic defects underlying many familial cases remain still unknown. Considering the high prevalence of PA in hypertensive patients and the current criteria for defining familial forms, a certain number of familial cases may however be due to co-occurrence of sporadic PA within the same family. Further studies will need to address whether other genes are involved in APA and familial hyperaldosteronism and replicate genetic findings observed in bilateral forms of the disease.

Summary

Understanding the genetic basis of primary aldosteronism is important to improve diagnosis and therapeutic approaches of the disease. During the last ten years, whole exome sequencing has been successful applied to identify the genetic determinants of PA. Mutations in genes coding for ion channels and pumps have been identified in APA as well as in familial hyperaldosteronism, all affecting intracellular ion homeostasis. Moreover, the recent identification of mutations in APA driver genes in APCC constitutes a new path to understand the mechanisms responsible for bilateral adrenal hyperplasia and opens new perspectives for therapeutic approaches in PA.

Practice points

 Somatic mutations in genes regulating intracellular ion homeostasis are present in at least 90% of APA.

 APCC harboring somatic mutations in APA driver genes may contribute to APA development and to bilateral adrenal hyperplasia  Genetic discoveries in PA have the potential to improve diagnosis and treatment of PA in the near future.

Research agenda

 Whole genome sequencing and characterization of the epigenome to further decipher the genetic basis of PA, particularly for BAH.

Figure 1 :

 1 Figure 1: Steroidogenic pathway for aldosterone and cortisol production in the adrenal

Figure 2 :

 2 Figure 2: Mechanisms responsible for Familial Hyperaldosteronism. (A) FH-I is due to

Table 1 .

 1 Frequency of somatic mutations in Aldosterone Producing Adenomas

	1
	2
	Mutation	Sanger Sequencing

 Development of new methods allowing preoperative detection of somatic mutations to improve the diagnosis and treatment of patients with PA.

 Need of functional studies to decipher the sequence of events leading to APA formation.

Disclosure statement

The authors declare that none have either any financial interest or conflicts of interest.