Genetic Characterization of GnRH/LH-Responsive Primary Aldosteronism

Genetic Characterization of GnRH/LH-Responsive Primary Aldosteronism Abstract Background Recently, somatic β-catenin mutations (CTNNB1) identified in aldosterone-producing adenomas (APAs) from three women were suggested to be responsible for the aberrant overexpression of luteinizing hormone/choriogonadotropin receptor and gonadotropin-releasing hormone receptor in the APA. Objective To genetically characterize patients with primary aldosteronism (PA) evaluated in vivo for gonadotropin-releasing hormone (GnRH)/luteinizing hormone (LH)-responsive aldosterone secretion. Method Patients with PA were evaluated in vivo to determine the possible regulation of aldosterone secretion by GnRH or LH. Genetic analysis of the CTNNB1, KCNJ5, ATP1A1, ATP2B3, CACNA1D, and GNAS genes were performed in this cohort and a control cohort of PA not tested in vivo for GnRH response. Results We studied 50 patients with confirmed PA, including 36 APAs, 12 bilateral macronodular adrenal hyperplasias, 1 oncocytoma, and 1 bilateral hyperplasia with cosecretion of cortisol. Among 23 patients tested in vivo for GnRH response of aldosterone, 7 (30.4%) had a positive response, 4 (17.4%) a partial response, and 12 (52.2%) no response. No somatic CTNNB1 mutations were identified, but the disease-causing c.451G>C KCNJ5 mutation was found in two individuals with partial and no GnRH responses and an individual showing a positive response to LH. Two additional somatic pathogenic mutations, CACNA1D c.776T>A and ATP1A1 c.311T>G, were identified in two patients with no GnRH responses. In the 26 patients not tested for GnRH response, we identified 2 CTNNB1 (7.7%), 13 KCNJ5 (50%), and 1 CACNA1D (3.8%) mutations. Conclusion Aberrant regulation of aldosterone by GnRH is frequent in PA, but is not often associated with somatic CTNNB1, although it may be found with somatic KCNJ5 mutations. Primary aldosteronism (PA) is a frequent cause of secondary hypertension, estimated to affect 5% to 10% of the general hypertensive population and up to 20% of patients with treatment-resistant hypertension (1–3). Aldosterone-producing adenoma (APA) is one of the most common (30% to 50%) form with bilateral adrenal zona glomerulosa hyperplasia (50% to 70%) (4). The mechanisms implicated in the pathophysiology of PA are not fully elucidated. Recently, somatic mutations with gain of function in the potassium channel gene KCNJ5 were found in 30%–40% of APA (5–8). Mutations with loss of function in ATP1A1 and ATP2B3 genes were identified in 5.2% and 1.6%, respectively, of patients with APA (9). Somatic and germline mutations with gain of function in the CACNA1D gene, encoding a voltage-gated calcium channel, were described in 7.8% in APA (10). Four germline CACNA1H mutations in patients with PA with different phenotypic presentations were identified (11). The WNT signaling pathway is important for normal development of the adrenal cortex and specifically the zona glomerulosa (12). Activation of the Wnt/β-catenin pathway was previously described in APA (13–15). However, somatic mutations in the CTNNB1 gene coding for β-catenin were identified with a low prevalence of 5.1% (10/196) and 3.7% (8/219) in APA (16, 17). Berthon et al. (15) provided evidence that decreased expression of a WNT inhibitor (SFRP2) may contribute to deregulate WNT signaling. They also demonstrate that β-catenin plays an essential role in the basal and Angiotensin II-induced aldosterone secretion (15). In 2011, Albiger et al. (18) described a case of PA diagnosed during pregnancy, and the authors suggested that, in a subset of patients with PA, the aberrant expression of luteinizing hormone receptor and gonadotropin-releasing hormone receptor (GNRHR) could modulate aldosterone secretion. More recently, somatic-activating mutations of CTNNB1 were identified in APA from two pregnant women and one postmenopausal woman, and the authors concluded that the CTNNB1 mutations may lead to the aberrant overexpression of luteinizing hormone/choriogonadotropin receptor (LHCGR) and GNRHR in the APA (19). We report here a cohort of patients with PA that were evaluated in vivo for the presence of gonadotropin-releasing hormone (GnRH) response in aldosterone. In addition, we describe genetic data, including CTNNB1, GNAS, KCNJ5, ATP1A1, ATP2B3 and CACNA1D genes genetic analysis in their adrenocortical tissues. We show that CTNNB1 gene mutations are not frequent in GnRH-responsive PA, but mutations in the KCNJ5 gene may be found. Methods Patients and tissue collection We studied a cohort of 50 patients with confirmed PA who underwent unilateral adrenalectomy at Centre hospitalier de l’Université de Montréal between 2004 and 2017. The institutional ethics committee approved the investigation protocol, and every participant provided an informed consent. Data, including patient age, sex, presence of high blood pressure and hypokalemia, results of biochemical and imaging studies were collected. Data concerning cortisol cosecretion were also collected, including a 1 mg overnight dexamethasone suppression test. The diagnosis of PA was in accordance with the guidelines of the Endocrine Society, with an aldosterone-to-renin ratio (ARR) >550 pmol/L:ng/mL/h, and was confirmed by elevated 24-hour urinary aldosterone secretion (>38 nmol/d) following 3 days of high oral salt intake or abnormal aldosterone suppression following 2 L of 0.9% saline IV over 4 hours in a seated position (>169 pmol/L) (20). When available, data of adrenal venous sampling were collected. Aberrant G-protein coupled receptor protocol Before surgery, a subgroup of patients was evaluated with an in vivo clinical protocol to determine the possible regulation of aldosterone secretion modulated by the adrenocortical aberrant expression of various G-protein coupled receptors (21, 22). To alleviate the effect of adrenocorticotropin hormone on aldosterone production, 1 mg of dexamethasone was administered orally every 6 hours, 2 days before the beginning of the protocol, and was continued for 3 days during the testing. All tests were performed fasting with the patient in supine position for at least 60 minutes. As previously described, the clinical protocol consisted of plasma level measurements of aldosterone, renin, cortisol, and adrenocorticotropin hormone at 30- to 60-minute intervals for 2 to 3 hours during tests that transiently modulate the levels of ligands for potential aberrant receptors. The protocol included administration of 100 mcg GnRH IV (Factrel; Wyeth-Ayerst, Montréal, Quebec, Canada) and in a subset of patients was completed by the IV injection of 300 IU of luteinizing hormone (LH) (LHadi; Serono Canada, Oakville, Ontario, Canada). A positive response was defined as a >50% renin-independent increase in aldosterone following GnRH administration. A partial response was described as increase in aldosterone between 25% and 50%, and no response if the increase was <25%. Mutational analysis Leukocyte DNA and tumoral DNA were extracted after adrenalectomy from fresh frozen tissues as described previously (23). Specific exons of the coding regions were amplified by PCR for the following genes: CTNNB1 (exon 3), KCNJ5 (exon 2), GNAS (exons 8 and 9), ATP1A1 (exons 4 and 8), ATP2B3 (exon 8), and CACNA1D (exons 6, 8, 14, 16, 23, 27, and 33). The amplicons were directly sequenced using the Applied Biosystems 3730xl DNA Analyzer (McGill University and Genome Quebec Innovation Centre, Quebec, Canada). All the primers that were used are described in Supplemental Table 1. RT-PCR expression of GNRHR and LHCGR Total RNA of adrenal tissues was extracted with TRIzol reagent (Invitrogen, Carlsbad, CA) from 12 patients who were tested in vivo for the presence of aldosterone response to GnRH and five patients for aldosterone response to LH. A commercially available pool of human adrenal total RNA (Clontech, Mountain View, CA) was used as reference sample. Quantitative RT-PCR was performed in the Quant Studio 6flex system using TaqMan gene expression assays (Applied Biosystem, Foster City, CA). To make a choice for endogenous control, we used the TaqMan Array Human Endogenous Controls Plate (4396840; Life Technologies, Carlsbad, CA). The assay IDs were Hs00171248_m1 for the GNRHR gene and Hs00896336_m1 for the LHCGR gene. A cycle threshold value in the linear range of amplification was selected for each sample in triplicate and normalized to human α growth arrest and DNA damage inducible (GADD45A) endogenous control gene (Hs00169255_m1). Relative quantification was performed using the 2–ΔΔCT method. Graphs and statistical analyses were made using GraphPad Prism software version 7.0 (La Jolla, CA). Student t test was used for two-group comparisons and one-way ANOVA for three-group comparisons. P values <0.05 were considered to be significant. Western blotting and immunohistochemistry analysis of β-catenin protein All protein extractions from seven adrenal tissues were probed with rabbit anti-nonphospho (active) β-catenin (1:1000) (mAb#8814; New England Biolabs, Ipswich, MA) and mouse antiactin (1:2500) (ab3280, Clone ACTN05 (C4); Abcam, Cambridge, MA) antibodies. Each primary antibody was detected with an anti-rabbit or anti-mouse horseradish peroxidase (Bio-Rad, Hercules, CA) at 1:3000 for 1 hour and developed with enhanced chemiluminescence Western blotting detection reagent (Thermo Fisher Scientific, Waltham, PA). Images were obtained from a ChemiDoc MP Imaging System (Bio-Rad), and densitometric analysis was made using Image Laboratory software version 6.0 (Bio-Rad). Actin was used as loading control, and a pooled homogenate from all of the samples was used as control sample. Immunohistochemical analysis was also conducted in the tissue of a patient harboring a β-catenin mutation. Original and new hematoxylin and eosin slides of the APA tumor were reviewed by an experienced pathologist (M.L.). β-Catenin was performed on 3-mm-thick sections of deparaffinized tissue, and antigens were retrieved. The slides were incubated with mouse monoclonal antibodies (Ventana BenchMark System, Tucson, AZ) against β-catenin (clone β-catenin 1, 1:200 dilution) (Dako, Santa Clara, CA). External positive controls were performed. Staining was scored semiquantitatively as 0 (negative), 1+ (focally or weakly positive), 2+ (moderate staining), 3+ (diffuse strong staining), or 4+ (intense diffuse staining). Results Clinical and biochemical characteristics of all cohort of patients studied with PA The clinical and pathological characteristics of the 50 patients with confirmed diagnosis of PA are presented in Table 1. This cohort included 24 males (48%) and 26 females (52%) aged between 28 and 78 years old (mean: 52 year). Thirty were French Canadian (60%), 2 were English Canadian (4%), and 18 were from other ethnic origins (36%). Clinical presentation was hypertension alone in seven cases (14%) and hypertension with hypokalemia in 43 cases (86%). The ARR was available in 49 cases and was >550 in 44 cases (90%). Based on the absence of suppression after the administration of 1 mg dexamethasone at night, 13 (26%) had at least modest cosecretion of cortisol. Adrenal imaging was performed with CT scan in 44 cases (88%) and/or MRI in 22 cases (44%). The adrenal venous sampling was completed in 43 cases (86%), and 39 APA (91%) had a lateralization index on the side of the adenoma. Table 1. Clinical and Pathological Characteristics of Patients Patient Number Sex/Age ARR (N < 550) (pmol/L:ng/mL/h) Serum K+ (mmol/L) Pathological Diagnosis Secretion Side (R/L)/Size (cm) 1 F/40 4030 3.4 APA Aldo L/1.7 2 M/38 3180 3.6 APA Aldo R/0.5 3 F/55 192 4.6 BMAH Aldo L/1.4 4 M/51 4425 3.2 BMAH Aldo R/1.1 5 F/52 1552 3.3 APA Aldo L/0.8 6 M/67 7795 3.9 BMAH Aldo R/1.5 7 F/66 N/A APA Aldo and cortisol R/1.2 8 M/42 1126 2.9 APA Aldo R/1.2 9 F/52 339 (pmol/L:pg/mL) 2.9 APA Aldo R/2.0 10 M/45 2163 2.9 APA Aldo L/0.8 11 M/47 7255 3.2 APA Aldo L/1.0 12 M/50 6500 2.8 BMAH Aldo L/1.3 13 F/38 8387 2.1 APA Aldo and cortisol R/1.3 14 F/32 5780 3.3 BMAH Aldo L/1.7 15 F/49 3145 3.2 BMAH Aldo and cortisol R/2.2 16 F/52 7260 3.3 APA Aldo and cortisol L/2.2 17 M/53 4043 4.5 BMAH Aldo R/1.2 18 M/53 320 3.2 APA Aldo and cortisol L/1.4 19 F/60 2828 3.4 BMAH Aldo and cortisol R/4.0 20 F/33 6460 3.3 APA Aldo L/1.8 21 F/34 3868 2.9 APA Aldo L/2.3 22 F/48 291 2.8 ACC and APA Aldo and cortisol D/2.0 23 M/72 1035 3.3 APA Aldo L/1.2 24 F/61 4063 3.9 APA Aldo L/1.0 25 M/54 2285 3.4 APA Aldo L/0.8 26 M/35 4570 3.9 APA Aldo L/1.5 27 F/71 5440 3.2 APA Aldo L/1.6 28 F/61 91 3.6 Oncocytoma Aldo and cortisol R/10.5 29 M/60 650 4.3 APA Aldo R/1.2 30 M/55 3289 3.4 APA Aldo L/0.9 31 F/54 134 4.5 BMAH Aldo and cortisol L/2.3 32 F/55 792 2.8 APA Aldo L/1.7 33 M/64 4322 4.3 APA Aldo L/0.9 34 F/69 18,341 3.7 APA Aldo R/3.0 35 M/51 9040 4.3 APA Aldo L/1.9 36 M/57 11,090 3.3 APA Aldo L/1.0 37 F/38 1060 3.1 APA Aldo L/1.3 38 F/59 4905 1.9 APA Aldo L/1.5 39 M/56 984 4.3 APA Aldo R/2.5 40 F/38 15,367 3.3 APA Aldo R/1.6 41 M/64 2513 3.1 APA Aldo L/0.5 42 F/43 297 3.4 IHA and cortisol-secreting adenoma Aldo and cortisol L/3.0 43 F/56 1180 3.2 APA Aldo L/0.8 44 F/28 2680 3.4 BMAH Aldo L/N/A 45 F/63 5550 3.3 BMAH Aldo and cortisol R/3.5 46 M/38 3185 3.4 APA Aldo R/1.3 47 M/72 5220 3.1 APA Aldo and cortisol R/1.0 48 M/38 2005 3.3 BMAH Aldo and cortisol L/10.0 49 M/53 5115 3.1 APA Aldo R/0.6 50 M/78 9025 1.9 APA Aldo L/2.0 Patient Number Sex/Age ARR (N < 550) (pmol/L:ng/mL/h) Serum K+ (mmol/L) Pathological Diagnosis Secretion Side (R/L)/Size (cm) 1 F/40 4030 3.4 APA Aldo L/1.7 2 M/38 3180 3.6 APA Aldo R/0.5 3 F/55 192 4.6 BMAH Aldo L/1.4 4 M/51 4425 3.2 BMAH Aldo R/1.1 5 F/52 1552 3.3 APA Aldo L/0.8 6 M/67 7795 3.9 BMAH Aldo R/1.5 7 F/66 N/A APA Aldo and cortisol R/1.2 8 M/42 1126 2.9 APA Aldo R/1.2 9 F/52 339 (pmol/L:pg/mL) 2.9 APA Aldo R/2.0 10 M/45 2163 2.9 APA Aldo L/0.8 11 M/47 7255 3.2 APA Aldo L/1.0 12 M/50 6500 2.8 BMAH Aldo L/1.3 13 F/38 8387 2.1 APA Aldo and cortisol R/1.3 14 F/32 5780 3.3 BMAH Aldo L/1.7 15 F/49 3145 3.2 BMAH Aldo and cortisol R/2.2 16 F/52 7260 3.3 APA Aldo and cortisol L/2.2 17 M/53 4043 4.5 BMAH Aldo R/1.2 18 M/53 320 3.2 APA Aldo and cortisol L/1.4 19 F/60 2828 3.4 BMAH Aldo and cortisol R/4.0 20 F/33 6460 3.3 APA Aldo L/1.8 21 F/34 3868 2.9 APA Aldo L/2.3 22 F/48 291 2.8 ACC and APA Aldo and cortisol D/2.0 23 M/72 1035 3.3 APA Aldo L/1.2 24 F/61 4063 3.9 APA Aldo L/1.0 25 M/54 2285 3.4 APA Aldo L/0.8 26 M/35 4570 3.9 APA Aldo L/1.5 27 F/71 5440 3.2 APA Aldo L/1.6 28 F/61 91 3.6 Oncocytoma Aldo and cortisol R/10.5 29 M/60 650 4.3 APA Aldo R/1.2 30 M/55 3289 3.4 APA Aldo L/0.9 31 F/54 134 4.5 BMAH Aldo and cortisol L/2.3 32 F/55 792 2.8 APA Aldo L/1.7 33 M/64 4322 4.3 APA Aldo L/0.9 34 F/69 18,341 3.7 APA Aldo R/3.0 35 M/51 9040 4.3 APA Aldo L/1.9 36 M/57 11,090 3.3 APA Aldo L/1.0 37 F/38 1060 3.1 APA Aldo L/1.3 38 F/59 4905 1.9 APA Aldo L/1.5 39 M/56 984 4.3 APA Aldo R/2.5 40 F/38 15,367 3.3 APA Aldo R/1.6 41 M/64 2513 3.1 APA Aldo L/0.5 42 F/43 297 3.4 IHA and cortisol-secreting adenoma Aldo and cortisol L/3.0 43 F/56 1180 3.2 APA Aldo L/0.8 44 F/28 2680 3.4 BMAH Aldo L/N/A 45 F/63 5550 3.3 BMAH Aldo and cortisol R/3.5 46 M/38 3185 3.4 APA Aldo R/1.3 47 M/72 5220 3.1 APA Aldo and cortisol R/1.0 48 M/38 2005 3.3 BMAH Aldo and cortisol L/10.0 49 M/53 5115 3.1 APA Aldo R/0.6 50 M/78 9025 1.9 APA Aldo L/2.0 Abbreviations: Aldo, aldosterone; F, female; L, left; M, male; N/A, not available; R, right. View Large Table 1. Clinical and Pathological Characteristics of Patients Patient Number Sex/Age ARR (N < 550) (pmol/L:ng/mL/h) Serum K+ (mmol/L) Pathological Diagnosis Secretion Side (R/L)/Size (cm) 1 F/40 4030 3.4 APA Aldo L/1.7 2 M/38 3180 3.6 APA Aldo R/0.5 3 F/55 192 4.6 BMAH Aldo L/1.4 4 M/51 4425 3.2 BMAH Aldo R/1.1 5 F/52 1552 3.3 APA Aldo L/0.8 6 M/67 7795 3.9 BMAH Aldo R/1.5 7 F/66 N/A APA Aldo and cortisol R/1.2 8 M/42 1126 2.9 APA Aldo R/1.2 9 F/52 339 (pmol/L:pg/mL) 2.9 APA Aldo R/2.0 10 M/45 2163 2.9 APA Aldo L/0.8 11 M/47 7255 3.2 APA Aldo L/1.0 12 M/50 6500 2.8 BMAH Aldo L/1.3 13 F/38 8387 2.1 APA Aldo and cortisol R/1.3 14 F/32 5780 3.3 BMAH Aldo L/1.7 15 F/49 3145 3.2 BMAH Aldo and cortisol R/2.2 16 F/52 7260 3.3 APA Aldo and cortisol L/2.2 17 M/53 4043 4.5 BMAH Aldo R/1.2 18 M/53 320 3.2 APA Aldo and cortisol L/1.4 19 F/60 2828 3.4 BMAH Aldo and cortisol R/4.0 20 F/33 6460 3.3 APA Aldo L/1.8 21 F/34 3868 2.9 APA Aldo L/2.3 22 F/48 291 2.8 ACC and APA Aldo and cortisol D/2.0 23 M/72 1035 3.3 APA Aldo L/1.2 24 F/61 4063 3.9 APA Aldo L/1.0 25 M/54 2285 3.4 APA Aldo L/0.8 26 M/35 4570 3.9 APA Aldo L/1.5 27 F/71 5440 3.2 APA Aldo L/1.6 28 F/61 91 3.6 Oncocytoma Aldo and cortisol R/10.5 29 M/60 650 4.3 APA Aldo R/1.2 30 M/55 3289 3.4 APA Aldo L/0.9 31 F/54 134 4.5 BMAH Aldo and cortisol L/2.3 32 F/55 792 2.8 APA Aldo L/1.7 33 M/64 4322 4.3 APA Aldo L/0.9 34 F/69 18,341 3.7 APA Aldo R/3.0 35 M/51 9040 4.3 APA Aldo L/1.9 36 M/57 11,090 3.3 APA Aldo L/1.0 37 F/38 1060 3.1 APA Aldo L/1.3 38 F/59 4905 1.9 APA Aldo L/1.5 39 M/56 984 4.3 APA Aldo R/2.5 40 F/38 15,367 3.3 APA Aldo R/1.6 41 M/64 2513 3.1 APA Aldo L/0.5 42 F/43 297 3.4 IHA and cortisol-secreting adenoma Aldo and cortisol L/3.0 43 F/56 1180 3.2 APA Aldo L/0.8 44 F/28 2680 3.4 BMAH Aldo L/N/A 45 F/63 5550 3.3 BMAH Aldo and cortisol R/3.5 46 M/38 3185 3.4 APA Aldo R/1.3 47 M/72 5220 3.1 APA Aldo and cortisol R/1.0 48 M/38 2005 3.3 BMAH Aldo and cortisol L/10.0 49 M/53 5115 3.1 APA Aldo R/0.6 50 M/78 9025 1.9 APA Aldo L/2.0 Patient Number Sex/Age ARR (N < 550) (pmol/L:ng/mL/h) Serum K+ (mmol/L) Pathological Diagnosis Secretion Side (R/L)/Size (cm) 1 F/40 4030 3.4 APA Aldo L/1.7 2 M/38 3180 3.6 APA Aldo R/0.5 3 F/55 192 4.6 BMAH Aldo L/1.4 4 M/51 4425 3.2 BMAH Aldo R/1.1 5 F/52 1552 3.3 APA Aldo L/0.8 6 M/67 7795 3.9 BMAH Aldo R/1.5 7 F/66 N/A APA Aldo and cortisol R/1.2 8 M/42 1126 2.9 APA Aldo R/1.2 9 F/52 339 (pmol/L:pg/mL) 2.9 APA Aldo R/2.0 10 M/45 2163 2.9 APA Aldo L/0.8 11 M/47 7255 3.2 APA Aldo L/1.0 12 M/50 6500 2.8 BMAH Aldo L/1.3 13 F/38 8387 2.1 APA Aldo and cortisol R/1.3 14 F/32 5780 3.3 BMAH Aldo L/1.7 15 F/49 3145 3.2 BMAH Aldo and cortisol R/2.2 16 F/52 7260 3.3 APA Aldo and cortisol L/2.2 17 M/53 4043 4.5 BMAH Aldo R/1.2 18 M/53 320 3.2 APA Aldo and cortisol L/1.4 19 F/60 2828 3.4 BMAH Aldo and cortisol R/4.0 20 F/33 6460 3.3 APA Aldo L/1.8 21 F/34 3868 2.9 APA Aldo L/2.3 22 F/48 291 2.8 ACC and APA Aldo and cortisol D/2.0 23 M/72 1035 3.3 APA Aldo L/1.2 24 F/61 4063 3.9 APA Aldo L/1.0 25 M/54 2285 3.4 APA Aldo L/0.8 26 M/35 4570 3.9 APA Aldo L/1.5 27 F/71 5440 3.2 APA Aldo L/1.6 28 F/61 91 3.6 Oncocytoma Aldo and cortisol R/10.5 29 M/60 650 4.3 APA Aldo R/1.2 30 M/55 3289 3.4 APA Aldo L/0.9 31 F/54 134 4.5 BMAH Aldo and cortisol L/2.3 32 F/55 792 2.8 APA Aldo L/1.7 33 M/64 4322 4.3 APA Aldo L/0.9 34 F/69 18,341 3.7 APA Aldo R/3.0 35 M/51 9040 4.3 APA Aldo L/1.9 36 M/57 11,090 3.3 APA Aldo L/1.0 37 F/38 1060 3.1 APA Aldo L/1.3 38 F/59 4905 1.9 APA Aldo L/1.5 39 M/56 984 4.3 APA Aldo R/2.5 40 F/38 15,367 3.3 APA Aldo R/1.6 41 M/64 2513 3.1 APA Aldo L/0.5 42 F/43 297 3.4 IHA and cortisol-secreting adenoma Aldo and cortisol L/3.0 43 F/56 1180 3.2 APA Aldo L/0.8 44 F/28 2680 3.4 BMAH Aldo L/N/A 45 F/63 5550 3.3 BMAH Aldo and cortisol R/3.5 46 M/38 3185 3.4 APA Aldo R/1.3 47 M/72 5220 3.1 APA Aldo and cortisol R/1.0 48 M/38 2005 3.3 BMAH Aldo and cortisol L/10.0 49 M/53 5115 3.1 APA Aldo R/0.6 50 M/78 9025 1.9 APA Aldo L/2.0 Abbreviations: Aldo, aldosterone; F, female; L, left; M, male; N/A, not available; R, right. View Large Pathological diagnosis was 35 APA (72%), 12 bilateral macronodular adrenal hyperplasia (BMAH) (24%), 1 bilateral hyperplasia (IHA) with a concomitant cortisol-secreting adenoma (2%), 1 oncocytoma (2%), and 1 adrenocortical carcinoma (ACC) with a concomitant APA (2%). Following adrenalectomy, follow-up for resolution of hypertension and/or hypokalemia was available in 45 cases (90%). Two cases showed resolution of the hypertension alone (4%), 14 cases had resolution of both the hypertension and the hypokalemia (31%), and 20 cases improved hypertension with resolution of hypokalemia (44%). Three cases persisted with hypertension alone (7%) and six cases with hypertension and hypokalemia (13%). Aldosterone response to stimulation with GnRH in vivo The in vivo stimulation with GnRH was performed in 23 patients, and a positive response (>50% renin-independent increase of aldosterone) was identified in seven patients (30.4%) (6 APAs and 1 BMAH), a partial response (>25% to 49% increase of aldosterone) in four patients (17.4%) (3 APAs and 1 IHA), and no response in 12 patients (52.2%) (7 APAs and 5 BMAHs) (Table 2). The in vivo stimulation with LH was performed in seven patients with GnRH response in aldosterone and showed a positive response (>50% renin-independent increase of aldosterone) in four patients (4 APAs) and no response in three patients (2 APAs and 1 IHA) (Table 2). Table 2. Responses to In Vivo Stimulation Test GnRH and/or LH and Genetic Analysis Patient Number Sex/Age Pathological Diagnosis Secretion GnRH (% of Aldosterone Elevation) LH (% of Aldosterone Elevation) CTNNB1 (Exon 3) GNAS (Exons 8 and 9) KCNJ5 (Exon 2) CACNA1D (Exons 6, 8A, 8B, 14, 16, 23, 27, and 33) ATP1A1 (Exons 4 and 8) ATP2B3 (Exon 8) 2 M/38 APA Aldo No response (–11%) N/A N/A N/A N/A N/A N/A N/A 3 F/55 BMAH Aldo No response (11%) N/A WT WT a WT WT WT 6 M/67 BMAH Aldo No response (–45%) N/A WT WT a WT WT WT 25 M/54 APA Aldo No response (–20%) N/A WT WT a WT c.311T>G, p.Leu104Argb WT 29 M/60 APA Aldo No response (–13%) N/A WT WT a WT WT WT 30 M/55 APA Aldo No response (18%) N/A WT WT c.837G>A, p.Glu279a,c c.776T>A, p.Val259Aspb WT WT 31 F/54 BMAH Aldo and cortisol No response (19%) N/A WT WT a WT WT WT 39 M/56 APA Aldo No response (3%) N/A WT WT a WT WT WT 40 F/38 APA Aldo No response (19%) No response (6%) WT WT c.451G>C, p.Gly151Arga,b WT WT WT 44 F/28 BMAH Aldo No response (18%) N/A WT WT a WT WT c.1224G>,
p.Pro408c 47 M/72 APA Aldo and cortisol No response (0%) N/A WT WT a WT WT WT 48 M/38 BMAH Aldo and cortisol No response (8%) N/A c.81A>G, p.Gln27c WT a WT WT WT 5 F/52 APA Aldo Partial response (47%) Positive response (100%) WT WT a WT WT WT 9 F/52 APA Aldo Partial response (42%) N/A WT WT a WT WT WT 26 M/35 APA Aldo Partial response (28%) N/A WT WT c.121C>T, p.Arg41Cysa,d c.451G>C, p.Gly151Argb WT WT WT 42 F/43 IHA Aldo and cortisol Partial response (42%) No response (N/A) WT c.601C>T, p.Arg201Cysb a WT WT WT 18 M/53 APA Aldo and cortisol Positive response (200%) Positive response (214%) WT WT a WT WT WT 24 F/61 APA Aldo Positive response (114%) N/A WT WT a WT WT WT 41 M/64 APA Aldo Positive response (118%) No response (9%) WT WT a WT WT WT 45 F/63 BMAH Aldo and cortisol Positive response (92%) N/A N/A N/A N/A N/A N/A N/A 46 M/38 APA Aldo Positive response (66%) Positive response (80%) WT WT a WT WT WT 49 M/53 APA Aldo Positive response (85%) N/A N/A N/A N/A N/A N/A N/A 50 M/78 APA Aldo Positive response (119%) Positive response (168%) WT WT a WT WT WT 21 F/34 APA Aldo N/A Positive response (65%) WT WT c.451G>C, p.Gly151Arga,b WT WT WT Patient Number Sex/Age Pathological Diagnosis Secretion GnRH (% of Aldosterone Elevation) LH (% of Aldosterone Elevation) CTNNB1 (Exon 3) GNAS (Exons 8 and 9) KCNJ5 (Exon 2) CACNA1D (Exons 6, 8A, 8B, 14, 16, 23, 27, and 33) ATP1A1 (Exons 4 and 8) ATP2B3 (Exon 8) 2 M/38 APA Aldo No response (–11%) N/A N/A N/A N/A N/A N/A N/A 3 F/55 BMAH Aldo No response (11%) N/A WT WT a WT WT WT 6 M/67 BMAH Aldo No response (–45%) N/A WT WT a WT WT WT 25 M/54 APA Aldo No response (–20%) N/A WT WT a WT c.311T>G, p.Leu104Argb WT 29 M/60 APA Aldo No response (–13%) N/A WT WT a WT WT WT 30 M/55 APA Aldo No response (18%) N/A WT WT c.837G>A, p.Glu279a,c c.776T>A, p.Val259Aspb WT WT 31 F/54 BMAH Aldo and cortisol No response (19%) N/A WT WT a WT WT WT 39 M/56 APA Aldo No response (3%) N/A WT WT a WT WT WT 40 F/38 APA Aldo No response (19%) No response (6%) WT WT c.451G>C, p.Gly151Arga,b WT WT WT 44 F/28 BMAH Aldo No response (18%) N/A WT WT a WT WT c.1224G>,
p.Pro408c 47 M/72 APA Aldo and cortisol No response (0%) N/A WT WT a WT WT WT 48 M/38 BMAH Aldo and cortisol No response (8%) N/A c.81A>G, p.Gln27c WT a WT WT WT 5 F/52 APA Aldo Partial response (47%) Positive response (100%) WT WT a WT WT WT 9 F/52 APA Aldo Partial response (42%) N/A WT WT a WT WT WT 26 M/35 APA Aldo Partial response (28%) N/A WT WT c.121C>T, p.Arg41Cysa,d c.451G>C, p.Gly151Argb WT WT WT 42 F/43 IHA Aldo and cortisol Partial response (42%) No response (N/A) WT c.601C>T, p.Arg201Cysb a WT WT WT 18 M/53 APA Aldo and cortisol Positive response (200%) Positive response (214%) WT WT a WT WT WT 24 F/61 APA Aldo Positive response (114%) N/A WT WT a WT WT WT 41 M/64 APA Aldo Positive response (118%) No response (9%) WT WT a WT WT WT 45 F/63 BMAH Aldo and cortisol Positive response (92%) N/A N/A N/A N/A N/A N/A N/A 46 M/38 APA Aldo Positive response (66%) Positive response (80%) WT WT a WT WT WT 49 M/53 APA Aldo Positive response (85%) N/A N/A N/A N/A N/A N/A N/A 50 M/78 APA Aldo Positive response (119%) Positive response (168%) WT WT a WT WT WT 21 F/34 APA Aldo N/A Positive response (65%) WT WT c.451G>C, p.Gly151Arga,b WT WT WT Positive response: elevation of aldosterone, >50%. Partial response: elevation of aldosterone, 25% to 50%. No response: elevation of aldosterone, <25%. Abbreviations: Aldo, aldosterone; F, female; M, male; N/A, not available; WT, wild-type. a Polymorphisms in KCNJ5 (c.171T>C, p.Ser57; c.810T>G, p.Leu270; and c.834T>C, p.His278). b Disease causing (pathogenic/likely pathogenic). c Polymorphism. d VUS. View Large Table 2. Responses to In Vivo Stimulation Test GnRH and/or LH and Genetic Analysis Patient Number Sex/Age Pathological Diagnosis Secretion GnRH (% of Aldosterone Elevation) LH (% of Aldosterone Elevation) CTNNB1 (Exon 3) GNAS (Exons 8 and 9) KCNJ5 (Exon 2) CACNA1D (Exons 6, 8A, 8B, 14, 16, 23, 27, and 33) ATP1A1 (Exons 4 and 8) ATP2B3 (Exon 8) 2 M/38 APA Aldo No response (–11%) N/A N/A N/A N/A N/A N/A N/A 3 F/55 BMAH Aldo No response (11%) N/A WT WT a WT WT WT 6 M/67 BMAH Aldo No response (–45%) N/A WT WT a WT WT WT 25 M/54 APA Aldo No response (–20%) N/A WT WT a WT c.311T>G, p.Leu104Argb WT 29 M/60 APA Aldo No response (–13%) N/A WT WT a WT WT WT 30 M/55 APA Aldo No response (18%) N/A WT WT c.837G>A, p.Glu279a,c c.776T>A, p.Val259Aspb WT WT 31 F/54 BMAH Aldo and cortisol No response (19%) N/A WT WT a WT WT WT 39 M/56 APA Aldo No response (3%) N/A WT WT a WT WT WT 40 F/38 APA Aldo No response (19%) No response (6%) WT WT c.451G>C, p.Gly151Arga,b WT WT WT 44 F/28 BMAH Aldo No response (18%) N/A WT WT a WT WT c.1224G>,
p.Pro408c 47 M/72 APA Aldo and cortisol No response (0%) N/A WT WT a WT WT WT 48 M/38 BMAH Aldo and cortisol No response (8%) N/A c.81A>G, p.Gln27c WT a WT WT WT 5 F/52 APA Aldo Partial response (47%) Positive response (100%) WT WT a WT WT WT 9 F/52 APA Aldo Partial response (42%) N/A WT WT a WT WT WT 26 M/35 APA Aldo Partial response (28%) N/A WT WT c.121C>T, p.Arg41Cysa,d c.451G>C, p.Gly151Argb WT WT WT 42 F/43 IHA Aldo and cortisol Partial response (42%) No response (N/A) WT c.601C>T, p.Arg201Cysb a WT WT WT 18 M/53 APA Aldo and cortisol Positive response (200%) Positive response (214%) WT WT a WT WT WT 24 F/61 APA Aldo Positive response (114%) N/A WT WT a WT WT WT 41 M/64 APA Aldo Positive response (118%) No response (9%) WT WT a WT WT WT 45 F/63 BMAH Aldo and cortisol Positive response (92%) N/A N/A N/A N/A N/A N/A N/A 46 M/38 APA Aldo Positive response (66%) Positive response (80%) WT WT a WT WT WT 49 M/53 APA Aldo Positive response (85%) N/A N/A N/A N/A N/A N/A N/A 50 M/78 APA Aldo Positive response (119%) Positive response (168%) WT WT a WT WT WT 21 F/34 APA Aldo N/A Positive response (65%) WT WT c.451G>C, p.Gly151Arga,b WT WT WT Patient Number Sex/Age Pathological Diagnosis Secretion GnRH (% of Aldosterone Elevation) LH (% of Aldosterone Elevation) CTNNB1 (Exon 3) GNAS (Exons 8 and 9) KCNJ5 (Exon 2) CACNA1D (Exons 6, 8A, 8B, 14, 16, 23, 27, and 33) ATP1A1 (Exons 4 and 8) ATP2B3 (Exon 8) 2 M/38 APA Aldo No response (–11%) N/A N/A N/A N/A N/A N/A N/A 3 F/55 BMAH Aldo No response (11%) N/A WT WT a WT WT WT 6 M/67 BMAH Aldo No response (–45%) N/A WT WT a WT WT WT 25 M/54 APA Aldo No response (–20%) N/A WT WT a WT c.311T>G, p.Leu104Argb WT 29 M/60 APA Aldo No response (–13%) N/A WT WT a WT WT WT 30 M/55 APA Aldo No response (18%) N/A WT WT c.837G>A, p.Glu279a,c c.776T>A, p.Val259Aspb WT WT 31 F/54 BMAH Aldo and cortisol No response (19%) N/A WT WT a WT WT WT 39 M/56 APA Aldo No response (3%) N/A WT WT a WT WT WT 40 F/38 APA Aldo No response (19%) No response (6%) WT WT c.451G>C, p.Gly151Arga,b WT WT WT 44 F/28 BMAH Aldo No response (18%) N/A WT WT a WT WT c.1224G>,
p.Pro408c 47 M/72 APA Aldo and cortisol No response (0%) N/A WT WT a WT WT WT 48 M/38 BMAH Aldo and cortisol No response (8%) N/A c.81A>G, p.Gln27c WT a WT WT WT 5 F/52 APA Aldo Partial response (47%) Positive response (100%) WT WT a WT WT WT 9 F/52 APA Aldo Partial response (42%) N/A WT WT a WT WT WT 26 M/35 APA Aldo Partial response (28%) N/A WT WT c.121C>T, p.Arg41Cysa,d c.451G>C, p.Gly151Argb WT WT WT 42 F/43 IHA Aldo and cortisol Partial response (42%) No response (N/A) WT c.601C>T, p.Arg201Cysb a WT WT WT 18 M/53 APA Aldo and cortisol Positive response (200%) Positive response (214%) WT WT a WT WT WT 24 F/61 APA Aldo Positive response (114%) N/A WT WT a WT WT WT 41 M/64 APA Aldo Positive response (118%) No response (9%) WT WT a WT WT WT 45 F/63 BMAH Aldo and cortisol Positive response (92%) N/A N/A N/A N/A N/A N/A N/A 46 M/38 APA Aldo Positive response (66%) Positive response (80%) WT WT a WT WT WT 49 M/53 APA Aldo Positive response (85%) N/A N/A N/A N/A N/A N/A N/A 50 M/78 APA Aldo Positive response (119%) Positive response (168%) WT WT a WT WT WT 21 F/34 APA Aldo N/A Positive response (65%) WT WT c.451G>C, p.Gly151Arga,b WT WT WT Positive response: elevation of aldosterone, >50%. Partial response: elevation of aldosterone, 25% to 50%. No response: elevation of aldosterone, <25%. Abbreviations: Aldo, aldosterone; F, female; M, male; N/A, not available; WT, wild-type. a Polymorphisms in KCNJ5 (c.171T>C, p.Ser57; c.810T>G, p.Leu270; and c.834T>C, p.His278). b Disease causing (pathogenic/likely pathogenic). c Polymorphism. d VUS. View Large Genetic analysis in the cohort of patients evaluated for aldosterone response to GnRH stimulation Among the 23 patients who underwent the in vivo stimulation with GnRH, we found no CTNNB1 mutations in the 11 patients with positive or partial response in aldosterone to GnRH. We found a silent CTNNB1 mutation, c.81A>G, p.Gln27, in a patient with aldosterone-secreting BMAH (patient 48) with no in vivo response to the GnRH. The c.601C>T, p.Arg201Cys GNAS mutation was found in the cortisol-secreting adenoma of a patient with concomitant IHA (patient 42), but no GNAS mutations were found in aldosterone-secreting tissues. This patient presented a partial response in aldosterone to GnRH and no response to LH stimulation. The somatic disease-causing KCNJ5 (c.451G>C, p.Gly151Arg) mutation was found in three patients with APA: One showed no response (patient 40), and one showed a positive response to LH stimulation, but was not tested with GnRH (patient 21). The third patient had a partial response (patient 26) of aldosterone to GnRH, and in addition to this disease-causing KCNJ5 somatic mutation, we identified in his leukocytes the germline KCNJ5 c.121C>T, p.Arg41Cys genetic alteration, which is a variant of uncertain significance (VUS). A ATP1A1 mutation (c.311T>G, p.Leu104Arg) (patient 25) and a CACNA1D mutation (c.776T>A, p.Val259Asp) (patient 30) were found in two patients (two APAs) with no response to in vivo stimulation with GnRH. Details of the responses to stimulation tests and various genetic alterations found are presented in Table 2 (Fig. 1; Supplemental Fig. 1). Figure 1. View largeDownload slide Sanger sequencing chromatograms showing somatic mutations p.Ser45Pro (CTNNB1), p.Arg201Cys (GNAS), p.Leu104Arg (ATP1A1), p.Gly151Arg (KCNJ5), p.Leu168Arg (KCNJ5), p.Val259Asp (CACNA1D), and p.Val1151Phe (CACNA1D). When the leukocyte DNA was not available, the adjacent normal tissue DNA was used. Figure 1. View largeDownload slide Sanger sequencing chromatograms showing somatic mutations p.Ser45Pro (CTNNB1), p.Arg201Cys (GNAS), p.Leu104Arg (ATP1A1), p.Gly151Arg (KCNJ5), p.Leu168Arg (KCNJ5), p.Val259Asp (CACNA1D), and p.Val1151Phe (CACNA1D). When the leukocyte DNA was not available, the adjacent normal tissue DNA was used. Genetic analysis in the cohort of patients not tested in vivo with GnRH stimulation Among the 26 patients who did not undergo the in vivo stimulation test with GnRH or LH, we found two pathogenic CTNNB1 mutations (c.133T>C, p.Ser45Pro): in a patient with BMAH cosecreting aldosterone and cortisol (patient 19) and 26 995 del 271 bp in one patient with APA (patient 34) (24). The somatic disease-causing KCNJ5 (c.451G>C, p.Gly151Arg) mutation was found in four patients (three APAs and one BMAH), and the somatic disease-causing KCNJ5 (c.451G>A, p.Gly151Arg) mutation was found in six patients (five APAs and one BMAH). The KCNJ5 mutation (c.503T>G, p.Leu168Arg) was found in one BMAH and two APAs. One CACNA1D mutation, c.3451G>T, p.Val1151Phe, was found in one patient with APA. No GNAS, ATP1A1, or ATP2B3 mutations were found. All details of genetic alterations found in the cohort of patients not tested in vivo with GnRH stimulation are presented in Table 3. Table 3. Genetic Analysis in Adrenal Tissues of Patients Without In Vivo Aberrant Receptor Stimulation Tests Patient Number Sex/Age Pathological Diagnosis Secretion CTNNB1 (Exon 3) GNAS (Exons 8 and 9) KCNJ5 (Exon 2) CACNA1D (Exons 6, 8A, 8B, 14, 16, 23, 27, and 33) ATP1A1 (Exons 4 and 8) ATP2B3 (Exon 8) 1 F/40 APA Aldo WT WT c.451G>A, p.Gly151Arga,b WT WT WT 4 M/51 BMAH Aldo WT WT a WT WT WT 7 F/66 APA Aldo and cortisol WT WT c.451G>C, p.Gly151Arga,b WT WT WT 8 M/42 APA Aldo WT WT c.224G>A, p.Ser75Asna,c WT WT WT 10 M/45 APA Aldo WT WT a WT WT WT 11 M/47 APA Aldo WT WT a WT WT WT 12 M/50 BMAH Aldo WT WT c.451G>C, p.Gly151Arga,b WT WT WT 13 F/38 APA Aldo and cortisol WT WT a WT WT WT 14 F/32 BMAH Aldo WT WT c.503T>G, p.Leu168Arga,b WT WT WT 15 F/49 BMAH Aldo and cortisol WT WT c.451G>A,p.Gly151Arga,b WT WT WT 16 F/52 APA Aldo and cortisol WT WT c.451G>A, p.Gly151Arga,b WT WT WT 17 M/53 BMAH Aldo WT WT a WT WT WT 19 F/60 BMAH Aldo and cortisol c.133T>C, p.Ser45Prob WT a WT WT WT 20 F/33 APA Aldo WT WT c.503T>G, p.Leu168Argb WT WT WT 22 F/48 ACC and APA Aldo and cortisol WT WT c.451G>A, p.Gly151Arga,b WT WT WT 23 M/72 APA Aldo WT WT a WT WT WT 27 F/71 APA Aldo WT WT c.503T>G, p.Leu168Arga,b WT WT WT 28 F/61 Oncocytoma Aldo and cortisol WT WT a WT WT WT 32 F/55 APA Aldo WT WT c.451G>C, p.Gly151Arga,b WT WT WT 33 M/64 APA Aldo WT WT a WT WT WT 34 F/69 APA Aldo 26 995 del 271 bpb p.Ala5_Ala80del WT a WT WT WT 35 M/51 APA Aldo WT WT c.451G>C, p.Gly151Arga,b WT WT WT 36 M/57 APA Aldo WT WT a WT WT WT 37 F/38 APA Aldo WT WT a c.3451G>T, p.Val1151Pheb WT WT 38 F/59 APA Aldo WT WT c.451G>A, p.Gly151Arga,b WT WT WT 43 F/56 APA Aldo WT WT c.451G>A, p.Gly151Arga,b WT WT WT Patient Number Sex/Age Pathological Diagnosis Secretion CTNNB1 (Exon 3) GNAS (Exons 8 and 9) KCNJ5 (Exon 2) CACNA1D (Exons 6, 8A, 8B, 14, 16, 23, 27, and 33) ATP1A1 (Exons 4 and 8) ATP2B3 (Exon 8) 1 F/40 APA Aldo WT WT c.451G>A, p.Gly151Arga,b WT WT WT 4 M/51 BMAH Aldo WT WT a WT WT WT 7 F/66 APA Aldo and cortisol WT WT c.451G>C, p.Gly151Arga,b WT WT WT 8 M/42 APA Aldo WT WT c.224G>A, p.Ser75Asna,c WT WT WT 10 M/45 APA Aldo WT WT a WT WT WT 11 M/47 APA Aldo WT WT a WT WT WT 12 M/50 BMAH Aldo WT WT c.451G>C, p.Gly151Arga,b WT WT WT 13 F/38 APA Aldo and cortisol WT WT a WT WT WT 14 F/32 BMAH Aldo WT WT c.503T>G, p.Leu168Arga,b WT WT WT 15 F/49 BMAH Aldo and cortisol WT WT c.451G>A,p.Gly151Arga,b WT WT WT 16 F/52 APA Aldo and cortisol WT WT c.451G>A, p.Gly151Arga,b WT WT WT 17 M/53 BMAH Aldo WT WT a WT WT WT 19 F/60 BMAH Aldo and cortisol c.133T>C, p.Ser45Prob WT a WT WT WT 20 F/33 APA Aldo WT WT c.503T>G, p.Leu168Argb WT WT WT 22 F/48 ACC and APA Aldo and cortisol WT WT c.451G>A, p.Gly151Arga,b WT WT WT 23 M/72 APA Aldo WT WT a WT WT WT 27 F/71 APA Aldo WT WT c.503T>G, p.Leu168Arga,b WT WT WT 28 F/61 Oncocytoma Aldo and cortisol WT WT a WT WT WT 32 F/55 APA Aldo WT WT c.451G>C, p.Gly151Arga,b WT WT WT 33 M/64 APA Aldo WT WT a WT WT WT 34 F/69 APA Aldo 26 995 del 271 bpb p.Ala5_Ala80del WT a WT WT WT 35 M/51 APA Aldo WT WT c.451G>C, p.Gly151Arga,b WT WT WT 36 M/57 APA Aldo WT WT a WT WT WT 37 F/38 APA Aldo WT WT a c.3451G>T, p.Val1151Pheb WT WT 38 F/59 APA Aldo WT WT c.451G>A, p.Gly151Arga,b WT WT WT 43 F/56 APA Aldo WT WT c.451G>A, p.Gly151Arga,b WT WT WT Abbreviations: Aldo, aldosterone; F, female; M, male; N/A, not available; WT, wild-type. a Polymorphisms in KCNJ5 (c.171T>C, p.Ser57; c.810T>G, p.Leu270; and c.834T>C, p.His278). b Disease causing (pathogenic/likely pathogenic). c VUS. View Large Table 3. Genetic Analysis in Adrenal Tissues of Patients Without In Vivo Aberrant Receptor Stimulation Tests Patient Number Sex/Age Pathological Diagnosis Secretion CTNNB1 (Exon 3) GNAS (Exons 8 and 9) KCNJ5 (Exon 2) CACNA1D (Exons 6, 8A, 8B, 14, 16, 23, 27, and 33) ATP1A1 (Exons 4 and 8) ATP2B3 (Exon 8) 1 F/40 APA Aldo WT WT c.451G>A, p.Gly151Arga,b WT WT WT 4 M/51 BMAH Aldo WT WT a WT WT WT 7 F/66 APA Aldo and cortisol WT WT c.451G>C, p.Gly151Arga,b WT WT WT 8 M/42 APA Aldo WT WT c.224G>A, p.Ser75Asna,c WT WT WT 10 M/45 APA Aldo WT WT a WT WT WT 11 M/47 APA Aldo WT WT a WT WT WT 12 M/50 BMAH Aldo WT WT c.451G>C, p.Gly151Arga,b WT WT WT 13 F/38 APA Aldo and cortisol WT WT a WT WT WT 14 F/32 BMAH Aldo WT WT c.503T>G, p.Leu168Arga,b WT WT WT 15 F/49 BMAH Aldo and cortisol WT WT c.451G>A,p.Gly151Arga,b WT WT WT 16 F/52 APA Aldo and cortisol WT WT c.451G>A, p.Gly151Arga,b WT WT WT 17 M/53 BMAH Aldo WT WT a WT WT WT 19 F/60 BMAH Aldo and cortisol c.133T>C, p.Ser45Prob WT a WT WT WT 20 F/33 APA Aldo WT WT c.503T>G, p.Leu168Argb WT WT WT 22 F/48 ACC and APA Aldo and cortisol WT WT c.451G>A, p.Gly151Arga,b WT WT WT 23 M/72 APA Aldo WT WT a WT WT WT 27 F/71 APA Aldo WT WT c.503T>G, p.Leu168Arga,b WT WT WT 28 F/61 Oncocytoma Aldo and cortisol WT WT a WT WT WT 32 F/55 APA Aldo WT WT c.451G>C, p.Gly151Arga,b WT WT WT 33 M/64 APA Aldo WT WT a WT WT WT 34 F/69 APA Aldo 26 995 del 271 bpb p.Ala5_Ala80del WT a WT WT WT 35 M/51 APA Aldo WT WT c.451G>C, p.Gly151Arga,b WT WT WT 36 M/57 APA Aldo WT WT a WT WT WT 37 F/38 APA Aldo WT WT a c.3451G>T, p.Val1151Pheb WT WT 38 F/59 APA Aldo WT WT c.451G>A, p.Gly151Arga,b WT WT WT 43 F/56 APA Aldo WT WT c.451G>A, p.Gly151Arga,b WT WT WT Patient Number Sex/Age Pathological Diagnosis Secretion CTNNB1 (Exon 3) GNAS (Exons 8 and 9) KCNJ5 (Exon 2) CACNA1D (Exons 6, 8A, 8B, 14, 16, 23, 27, and 33) ATP1A1 (Exons 4 and 8) ATP2B3 (Exon 8) 1 F/40 APA Aldo WT WT c.451G>A, p.Gly151Arga,b WT WT WT 4 M/51 BMAH Aldo WT WT a WT WT WT 7 F/66 APA Aldo and cortisol WT WT c.451G>C, p.Gly151Arga,b WT WT WT 8 M/42 APA Aldo WT WT c.224G>A, p.Ser75Asna,c WT WT WT 10 M/45 APA Aldo WT WT a WT WT WT 11 M/47 APA Aldo WT WT a WT WT WT 12 M/50 BMAH Aldo WT WT c.451G>C, p.Gly151Arga,b WT WT WT 13 F/38 APA Aldo and cortisol WT WT a WT WT WT 14 F/32 BMAH Aldo WT WT c.503T>G, p.Leu168Arga,b WT WT WT 15 F/49 BMAH Aldo and cortisol WT WT c.451G>A,p.Gly151Arga,b WT WT WT 16 F/52 APA Aldo and cortisol WT WT c.451G>A, p.Gly151Arga,b WT WT WT 17 M/53 BMAH Aldo WT WT a WT WT WT 19 F/60 BMAH Aldo and cortisol c.133T>C, p.Ser45Prob WT a WT WT WT 20 F/33 APA Aldo WT WT c.503T>G, p.Leu168Argb WT WT WT 22 F/48 ACC and APA Aldo and cortisol WT WT c.451G>A, p.Gly151Arga,b WT WT WT 23 M/72 APA Aldo WT WT a WT WT WT 27 F/71 APA Aldo WT WT c.503T>G, p.Leu168Arga,b WT WT WT 28 F/61 Oncocytoma Aldo and cortisol WT WT a WT WT WT 32 F/55 APA Aldo WT WT c.451G>C, p.Gly151Arga,b WT WT WT 33 M/64 APA Aldo WT WT a WT WT WT 34 F/69 APA Aldo 26 995 del 271 bpb p.Ala5_Ala80del WT a WT WT WT 35 M/51 APA Aldo WT WT c.451G>C, p.Gly151Arga,b WT WT WT 36 M/57 APA Aldo WT WT a WT WT WT 37 F/38 APA Aldo WT WT a c.3451G>T, p.Val1151Pheb WT WT 38 F/59 APA Aldo WT WT c.451G>A, p.Gly151Arga,b WT WT WT 43 F/56 APA Aldo WT WT c.451G>A, p.Gly151Arga,b WT WT WT Abbreviations: Aldo, aldosterone; F, female; M, male; N/A, not available; WT, wild-type. a Polymorphisms in KCNJ5 (c.171T>C, p.Ser57; c.810T>G, p.Leu270; and c.834T>C, p.His278). b Disease causing (pathogenic/likely pathogenic). c VUS. View Large Mutational analysis of the entire cohort of patients with PA Overall, two disease-causing mutations of the CTNNB1 gene were found among the 47 patients that were studied with PA (prevalence of 4.3%). One of 47 (2%) samples harbored a GNAS mutations (c.601C>T, p.Arg201Cys) in a cortisol-secreting adenoma with concomitant IHA. Somatic pathogenic/likely pathogenic KCNJ5 mutations were identified in 16/47 (34%) tissues studied, including c.451G>A, p.Gly151Arg (n = 6), c.451G>C, p.Gly151Arg (n = 7) (11 APAs and 2 BMAHs), and c.503T>G, p.Leu168Arg (n = 3) (2 APAs and 1 BMAH). In addition, two KCNJ5 germline VUSs, c.224G>A, p.Ser75Asn and c.121C>T, p.Arg41Cys, were identified in DNA leukocytes of two patients with APA. Two pathogenic CACNA1D mutations (4%), c.776T>A, p.Val259Asp in exon 6, and c.3451G>T, p.Val1151Phe in exon 27, were found in two APA samples. Moreover, a somatic pathogenic ATP1A1 mutation (2%) (c.311T>G, p.Leu104Arg) was identified in one APA. RT-PCR expression of GNRHR and LHCGR and Western blotting/immunohistochemical analysis of β-catenin protein As shown in Fig. 2, there was no significant statistical difference between mRNA expression of GNRHR and LHCGR in patients with positive, partial, or no response in aldosterone to GnRH in vivo. mRNA LHCGR expression was increased in two out of three patients with in vivo response to LH in aldosterone (Fig. 2), but not in patients with no response to LH. Western blotting analysis showed variable expression of β-catenin protein in patients with positive, partial, or no response to GnRH (Fig. 3). Immunohistochemical analysis of the adrenocortical tissue of patient 19 harboring a pathogenic CTNNB1 mutation showed diffuse, membranous staining for β-catenin (3+) and nuclear staining in isolated cells supporting activation of the Wnt/β-catenin signaling in this BMAH cosecreting cortisol and aldosterone. Figure 2. View largeDownload slide Relative mRNA expression of (A) GNRHR and (B) LHCGR in adrenal tissues from patients with PA: three with no response, three with partial response, and six with positive aberrant response of aldosterone to in vivo stimulation with GnRH. (C) Relative mRNA expression of LHCGR in a subgroup of the patients who were also tested for aldosterone response to LH administration: two with no response and three with positive response to LH. The relative quantification was performed by the 2–ΔΔCT method, using a pool of normal adrenal glands as calibrator and GADD45A as endogenous control gene. Each point represents a patient. GADD45A, human α growth arrest and DNA damage inducible. Figure 2. View largeDownload slide Relative mRNA expression of (A) GNRHR and (B) LHCGR in adrenal tissues from patients with PA: three with no response, three with partial response, and six with positive aberrant response of aldosterone to in vivo stimulation with GnRH. (C) Relative mRNA expression of LHCGR in a subgroup of the patients who were also tested for aldosterone response to LH administration: two with no response and three with positive response to LH. The relative quantification was performed by the 2–ΔΔCT method, using a pool of normal adrenal glands as calibrator and GADD45A as endogenous control gene. Each point represents a patient. GADD45A, human α growth arrest and DNA damage inducible. Figure 3. View largeDownload slide Western blot analysis of β-catenin protein in adrenal tissues from patients with PA: two with no response, two with partial response, and thre with positive response of aldosterone to in vivo stimulation with GnRH. Each point represents a patient. The values under the bands show results of densitometric analysis. They were normalized to actin and to control sample. The control (Ctl) sample is a pooled homogenate from all of the samples. Figure 3. View largeDownload slide Western blot analysis of β-catenin protein in adrenal tissues from patients with PA: two with no response, two with partial response, and thre with positive response of aldosterone to in vivo stimulation with GnRH. Each point represents a patient. The values under the bands show results of densitometric analysis. They were normalized to actin and to control sample. The control (Ctl) sample is a pooled homogenate from all of the samples. Discussion Our study combined the search for genetic alterations in six genes implicated in PA with in vivo aberrant regulation of aldosterone by GnRH and/or LH. Close to 50% of the 23 patients with PA increased their aldosterone following stimulation with GnRH or LH (four of seven responding to LH); previously Zwermann et al. (25) found a 25% response in 12 patients studied and Albiger et al. (18) found a 83% response in 12 patients (3 positive and 7 partial responses). The in vivo stimulation with GnRH was previously evaluated by 2 independent groups in 13 healthy subjects, and no increase in aldosterone was found in any (18, 25). We found two somatic disease-causing CTNNB1 mutations in two patients (one BMAH and one APA) who were not tested in vivo for a GnRH response. However, no CTNNB1 mutations were found among the 11 patients who had a positive or partial aberrant response of aldosterone to GnRH. Our findings are different from a previous report where CTNNB1 mutations were found in APA of two pregnant women and one postmenopausal woman, and a link was suggested between the CTNNB1 mutations and the aberrant expression of LHCGR and GNRHR (19). Another group described four women with CTNNB1 mutations in their APA, without association with pregnancy in any of the cases (26). In addition, a third group reported no CTNNB1 mutations among 26 APAs (27). In our cohort, the CTNNB1 mutations were found in two women of 60 and 69 years of age. Our sample size is too small to conclude to higher prevalence of CTNNB1 mutations in woman, neither at older age, as previously reported by others (16, 17). Unfortunately, the duration of hypertension before the surgery was unknown, but following surgery, complete remission occurred in two patients with a CTNNB1 mutation. A study reported a higher proportion of residual hypertension after adrenalectomy in patients with a CTNNB1 mutation, but this cannot be evaluated in our small sample size (17). Although, the activation of the WNT/β-catenin pathway is a major feature in APA, CTNNB1 mutations were identified in only 4% of our cohort (15). This is similar to two previous cohorts where a prevalence of 5.1% of somatic CTNNB1 mutations among 198 APAs and 3.7% among 209 APAs was found (16, 17). Thus, the prevalence of CTNNB1 mutations in APA remains much lower than nonsecreting or cortisol-secreting adrenocortical tumors (24, 28). The molecular mechanisms associated with CTNNB1 mutations in PA are still unclear. Recently, it was suggested that CTNNB1 mutations may be more related to tumorigenesis than to excessive aldosterone production (17, 29). In our cohort, CTNNB1 mutations occurred mutually exclusively to KCNJ5, GNAS, ATP1A1, ATP2B3, or CACNAD1 mutations. This is concordant with findings in two other cohorts of patients with APA (16, 17). We found an overall prevalence of 34% of pathogenic/likely pathogenic KCNJ5 mutations among our total cohort of 47 patients, which is similar to previous reports (5–8). Among those patients, one presented a partial response to stimulation with GnRH (patient 26) and another (patient 21) had a positive response to stimulation with LH. One study evaluated the expression of GNRHR and LHCGR in APA with (n = 13) and without (n = 9) KCNJ5 mutations (30). They found that GNRHR and LHCGR were highly expressed among APAs with no KCNJ5 mutations. Unfortunately, due to tissue availability, we studied a small number of adrenal tissues for GNRHR and LHCGR mRNA expression that showed variable expression not clearly correlated to the in vivo response to GnRH. In addition, according to the sites of the biopsy, results might be variables because of the heterogeneity of the adrenal tissues. We found a prevalence of 4% of CACNA1D mutations and 2% of ATP1A1 mutations. One of each was tested for GnRH response, and in both cases, there was no response to the in vivo GnRH stimulation. To our knowledge, no previous study has specifically looked at the expression of GNRHR in APA with CACNA1D, ATP1A1, or ATP2B3 mutations. There were strengths and limitations to our study. To our knowledge, this is the first study to compare the prevalence of six mutations implicated in PA with aberrant aldosterone stimulation by GnRH and/or LH in vivo. Unfortunately, not all patients were able to participate in the in vivo aberrant receptor screening protocol before their surgery. We found a similar prevalence of CTNNB1 mutations in our cohort as in previously published series. Further studies are needed to clarify the link between CTNNB1 mutations and development of PA. As previously suggested, it may be more related to tumorigenesis rather than to aldosterone production itself. Finally, CTNNB1 mutations do not lead to all in vivo GnRH positive or partial response in PA, as none of our patients with aberrant aldosterone regulation by GnRH or LH carried a somatic CTNNB1 mutation in their adrenocortical tissues. Abbreviations: Abbreviations: ACC adrenocortical carcinoma APA aldosterone-producing adenoma ARR aldosterone-to-renin ratio BMAH bilateral macronodular adrenal hyperplasia GnRH gonadotropin-releasing hormone GNRHR gonadotropin-releasing hormone receptor IHA bilateral hyperplasia LH luteinizing hormone LHCGR luteinizing hormone/choriogonadotropin receptor PA primary aldosteronism VUS variant of uncertain significance Acknowledgments Financial Support: This work was supported in part by a salary grant from Fonds de Recherche du Québec-Santé (to I.B.) and the Canadian Institutes of Health and Research (to I.B. and A.L.). Disclosure Summary: The authors have nothing to disclose. References 1. 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Kishimoto R , Oki K , Yoneda M , Gomez-Sanchez CE , Ohno H , Kobuke K , Itcho K , Kohno N . Gonadotropin-releasing hormone stimulate aldosterone production in a subset of aldosterone-producing adenoma . Medicine (Baltimore) . 2016 ; 95 ( 20 ): e3659 . Google Scholar CrossRef Search ADS PubMed Copyright © 2018 Endocrine Society http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Clinical Endocrinology and Metabolism Oxford University Press

Genetic Characterization of GnRH/LH-Responsive Primary Aldosteronism

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Oxford University Press
Copyright
Copyright © 2018 Endocrine Society
ISSN
0021-972X
eISSN
1945-7197
D.O.I.
10.1210/jc.2018-00087
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Abstract

Abstract Background Recently, somatic β-catenin mutations (CTNNB1) identified in aldosterone-producing adenomas (APAs) from three women were suggested to be responsible for the aberrant overexpression of luteinizing hormone/choriogonadotropin receptor and gonadotropin-releasing hormone receptor in the APA. Objective To genetically characterize patients with primary aldosteronism (PA) evaluated in vivo for gonadotropin-releasing hormone (GnRH)/luteinizing hormone (LH)-responsive aldosterone secretion. Method Patients with PA were evaluated in vivo to determine the possible regulation of aldosterone secretion by GnRH or LH. Genetic analysis of the CTNNB1, KCNJ5, ATP1A1, ATP2B3, CACNA1D, and GNAS genes were performed in this cohort and a control cohort of PA not tested in vivo for GnRH response. Results We studied 50 patients with confirmed PA, including 36 APAs, 12 bilateral macronodular adrenal hyperplasias, 1 oncocytoma, and 1 bilateral hyperplasia with cosecretion of cortisol. Among 23 patients tested in vivo for GnRH response of aldosterone, 7 (30.4%) had a positive response, 4 (17.4%) a partial response, and 12 (52.2%) no response. No somatic CTNNB1 mutations were identified, but the disease-causing c.451G>C KCNJ5 mutation was found in two individuals with partial and no GnRH responses and an individual showing a positive response to LH. Two additional somatic pathogenic mutations, CACNA1D c.776T>A and ATP1A1 c.311T>G, were identified in two patients with no GnRH responses. In the 26 patients not tested for GnRH response, we identified 2 CTNNB1 (7.7%), 13 KCNJ5 (50%), and 1 CACNA1D (3.8%) mutations. Conclusion Aberrant regulation of aldosterone by GnRH is frequent in PA, but is not often associated with somatic CTNNB1, although it may be found with somatic KCNJ5 mutations. Primary aldosteronism (PA) is a frequent cause of secondary hypertension, estimated to affect 5% to 10% of the general hypertensive population and up to 20% of patients with treatment-resistant hypertension (1–3). Aldosterone-producing adenoma (APA) is one of the most common (30% to 50%) form with bilateral adrenal zona glomerulosa hyperplasia (50% to 70%) (4). The mechanisms implicated in the pathophysiology of PA are not fully elucidated. Recently, somatic mutations with gain of function in the potassium channel gene KCNJ5 were found in 30%–40% of APA (5–8). Mutations with loss of function in ATP1A1 and ATP2B3 genes were identified in 5.2% and 1.6%, respectively, of patients with APA (9). Somatic and germline mutations with gain of function in the CACNA1D gene, encoding a voltage-gated calcium channel, were described in 7.8% in APA (10). Four germline CACNA1H mutations in patients with PA with different phenotypic presentations were identified (11). The WNT signaling pathway is important for normal development of the adrenal cortex and specifically the zona glomerulosa (12). Activation of the Wnt/β-catenin pathway was previously described in APA (13–15). However, somatic mutations in the CTNNB1 gene coding for β-catenin were identified with a low prevalence of 5.1% (10/196) and 3.7% (8/219) in APA (16, 17). Berthon et al. (15) provided evidence that decreased expression of a WNT inhibitor (SFRP2) may contribute to deregulate WNT signaling. They also demonstrate that β-catenin plays an essential role in the basal and Angiotensin II-induced aldosterone secretion (15). In 2011, Albiger et al. (18) described a case of PA diagnosed during pregnancy, and the authors suggested that, in a subset of patients with PA, the aberrant expression of luteinizing hormone receptor and gonadotropin-releasing hormone receptor (GNRHR) could modulate aldosterone secretion. More recently, somatic-activating mutations of CTNNB1 were identified in APA from two pregnant women and one postmenopausal woman, and the authors concluded that the CTNNB1 mutations may lead to the aberrant overexpression of luteinizing hormone/choriogonadotropin receptor (LHCGR) and GNRHR in the APA (19). We report here a cohort of patients with PA that were evaluated in vivo for the presence of gonadotropin-releasing hormone (GnRH) response in aldosterone. In addition, we describe genetic data, including CTNNB1, GNAS, KCNJ5, ATP1A1, ATP2B3 and CACNA1D genes genetic analysis in their adrenocortical tissues. We show that CTNNB1 gene mutations are not frequent in GnRH-responsive PA, but mutations in the KCNJ5 gene may be found. Methods Patients and tissue collection We studied a cohort of 50 patients with confirmed PA who underwent unilateral adrenalectomy at Centre hospitalier de l’Université de Montréal between 2004 and 2017. The institutional ethics committee approved the investigation protocol, and every participant provided an informed consent. Data, including patient age, sex, presence of high blood pressure and hypokalemia, results of biochemical and imaging studies were collected. Data concerning cortisol cosecretion were also collected, including a 1 mg overnight dexamethasone suppression test. The diagnosis of PA was in accordance with the guidelines of the Endocrine Society, with an aldosterone-to-renin ratio (ARR) >550 pmol/L:ng/mL/h, and was confirmed by elevated 24-hour urinary aldosterone secretion (>38 nmol/d) following 3 days of high oral salt intake or abnormal aldosterone suppression following 2 L of 0.9% saline IV over 4 hours in a seated position (>169 pmol/L) (20). When available, data of adrenal venous sampling were collected. Aberrant G-protein coupled receptor protocol Before surgery, a subgroup of patients was evaluated with an in vivo clinical protocol to determine the possible regulation of aldosterone secretion modulated by the adrenocortical aberrant expression of various G-protein coupled receptors (21, 22). To alleviate the effect of adrenocorticotropin hormone on aldosterone production, 1 mg of dexamethasone was administered orally every 6 hours, 2 days before the beginning of the protocol, and was continued for 3 days during the testing. All tests were performed fasting with the patient in supine position for at least 60 minutes. As previously described, the clinical protocol consisted of plasma level measurements of aldosterone, renin, cortisol, and adrenocorticotropin hormone at 30- to 60-minute intervals for 2 to 3 hours during tests that transiently modulate the levels of ligands for potential aberrant receptors. The protocol included administration of 100 mcg GnRH IV (Factrel; Wyeth-Ayerst, Montréal, Quebec, Canada) and in a subset of patients was completed by the IV injection of 300 IU of luteinizing hormone (LH) (LHadi; Serono Canada, Oakville, Ontario, Canada). A positive response was defined as a >50% renin-independent increase in aldosterone following GnRH administration. A partial response was described as increase in aldosterone between 25% and 50%, and no response if the increase was <25%. Mutational analysis Leukocyte DNA and tumoral DNA were extracted after adrenalectomy from fresh frozen tissues as described previously (23). Specific exons of the coding regions were amplified by PCR for the following genes: CTNNB1 (exon 3), KCNJ5 (exon 2), GNAS (exons 8 and 9), ATP1A1 (exons 4 and 8), ATP2B3 (exon 8), and CACNA1D (exons 6, 8, 14, 16, 23, 27, and 33). The amplicons were directly sequenced using the Applied Biosystems 3730xl DNA Analyzer (McGill University and Genome Quebec Innovation Centre, Quebec, Canada). All the primers that were used are described in Supplemental Table 1. RT-PCR expression of GNRHR and LHCGR Total RNA of adrenal tissues was extracted with TRIzol reagent (Invitrogen, Carlsbad, CA) from 12 patients who were tested in vivo for the presence of aldosterone response to GnRH and five patients for aldosterone response to LH. A commercially available pool of human adrenal total RNA (Clontech, Mountain View, CA) was used as reference sample. Quantitative RT-PCR was performed in the Quant Studio 6flex system using TaqMan gene expression assays (Applied Biosystem, Foster City, CA). To make a choice for endogenous control, we used the TaqMan Array Human Endogenous Controls Plate (4396840; Life Technologies, Carlsbad, CA). The assay IDs were Hs00171248_m1 for the GNRHR gene and Hs00896336_m1 for the LHCGR gene. A cycle threshold value in the linear range of amplification was selected for each sample in triplicate and normalized to human α growth arrest and DNA damage inducible (GADD45A) endogenous control gene (Hs00169255_m1). Relative quantification was performed using the 2–ΔΔCT method. Graphs and statistical analyses were made using GraphPad Prism software version 7.0 (La Jolla, CA). Student t test was used for two-group comparisons and one-way ANOVA for three-group comparisons. P values <0.05 were considered to be significant. Western blotting and immunohistochemistry analysis of β-catenin protein All protein extractions from seven adrenal tissues were probed with rabbit anti-nonphospho (active) β-catenin (1:1000) (mAb#8814; New England Biolabs, Ipswich, MA) and mouse antiactin (1:2500) (ab3280, Clone ACTN05 (C4); Abcam, Cambridge, MA) antibodies. Each primary antibody was detected with an anti-rabbit or anti-mouse horseradish peroxidase (Bio-Rad, Hercules, CA) at 1:3000 for 1 hour and developed with enhanced chemiluminescence Western blotting detection reagent (Thermo Fisher Scientific, Waltham, PA). Images were obtained from a ChemiDoc MP Imaging System (Bio-Rad), and densitometric analysis was made using Image Laboratory software version 6.0 (Bio-Rad). Actin was used as loading control, and a pooled homogenate from all of the samples was used as control sample. Immunohistochemical analysis was also conducted in the tissue of a patient harboring a β-catenin mutation. Original and new hematoxylin and eosin slides of the APA tumor were reviewed by an experienced pathologist (M.L.). β-Catenin was performed on 3-mm-thick sections of deparaffinized tissue, and antigens were retrieved. The slides were incubated with mouse monoclonal antibodies (Ventana BenchMark System, Tucson, AZ) against β-catenin (clone β-catenin 1, 1:200 dilution) (Dako, Santa Clara, CA). External positive controls were performed. Staining was scored semiquantitatively as 0 (negative), 1+ (focally or weakly positive), 2+ (moderate staining), 3+ (diffuse strong staining), or 4+ (intense diffuse staining). Results Clinical and biochemical characteristics of all cohort of patients studied with PA The clinical and pathological characteristics of the 50 patients with confirmed diagnosis of PA are presented in Table 1. This cohort included 24 males (48%) and 26 females (52%) aged between 28 and 78 years old (mean: 52 year). Thirty were French Canadian (60%), 2 were English Canadian (4%), and 18 were from other ethnic origins (36%). Clinical presentation was hypertension alone in seven cases (14%) and hypertension with hypokalemia in 43 cases (86%). The ARR was available in 49 cases and was >550 in 44 cases (90%). Based on the absence of suppression after the administration of 1 mg dexamethasone at night, 13 (26%) had at least modest cosecretion of cortisol. Adrenal imaging was performed with CT scan in 44 cases (88%) and/or MRI in 22 cases (44%). The adrenal venous sampling was completed in 43 cases (86%), and 39 APA (91%) had a lateralization index on the side of the adenoma. Table 1. Clinical and Pathological Characteristics of Patients Patient Number Sex/Age ARR (N < 550) (pmol/L:ng/mL/h) Serum K+ (mmol/L) Pathological Diagnosis Secretion Side (R/L)/Size (cm) 1 F/40 4030 3.4 APA Aldo L/1.7 2 M/38 3180 3.6 APA Aldo R/0.5 3 F/55 192 4.6 BMAH Aldo L/1.4 4 M/51 4425 3.2 BMAH Aldo R/1.1 5 F/52 1552 3.3 APA Aldo L/0.8 6 M/67 7795 3.9 BMAH Aldo R/1.5 7 F/66 N/A APA Aldo and cortisol R/1.2 8 M/42 1126 2.9 APA Aldo R/1.2 9 F/52 339 (pmol/L:pg/mL) 2.9 APA Aldo R/2.0 10 M/45 2163 2.9 APA Aldo L/0.8 11 M/47 7255 3.2 APA Aldo L/1.0 12 M/50 6500 2.8 BMAH Aldo L/1.3 13 F/38 8387 2.1 APA Aldo and cortisol R/1.3 14 F/32 5780 3.3 BMAH Aldo L/1.7 15 F/49 3145 3.2 BMAH Aldo and cortisol R/2.2 16 F/52 7260 3.3 APA Aldo and cortisol L/2.2 17 M/53 4043 4.5 BMAH Aldo R/1.2 18 M/53 320 3.2 APA Aldo and cortisol L/1.4 19 F/60 2828 3.4 BMAH Aldo and cortisol R/4.0 20 F/33 6460 3.3 APA Aldo L/1.8 21 F/34 3868 2.9 APA Aldo L/2.3 22 F/48 291 2.8 ACC and APA Aldo and cortisol D/2.0 23 M/72 1035 3.3 APA Aldo L/1.2 24 F/61 4063 3.9 APA Aldo L/1.0 25 M/54 2285 3.4 APA Aldo L/0.8 26 M/35 4570 3.9 APA Aldo L/1.5 27 F/71 5440 3.2 APA Aldo L/1.6 28 F/61 91 3.6 Oncocytoma Aldo and cortisol R/10.5 29 M/60 650 4.3 APA Aldo R/1.2 30 M/55 3289 3.4 APA Aldo L/0.9 31 F/54 134 4.5 BMAH Aldo and cortisol L/2.3 32 F/55 792 2.8 APA Aldo L/1.7 33 M/64 4322 4.3 APA Aldo L/0.9 34 F/69 18,341 3.7 APA Aldo R/3.0 35 M/51 9040 4.3 APA Aldo L/1.9 36 M/57 11,090 3.3 APA Aldo L/1.0 37 F/38 1060 3.1 APA Aldo L/1.3 38 F/59 4905 1.9 APA Aldo L/1.5 39 M/56 984 4.3 APA Aldo R/2.5 40 F/38 15,367 3.3 APA Aldo R/1.6 41 M/64 2513 3.1 APA Aldo L/0.5 42 F/43 297 3.4 IHA and cortisol-secreting adenoma Aldo and cortisol L/3.0 43 F/56 1180 3.2 APA Aldo L/0.8 44 F/28 2680 3.4 BMAH Aldo L/N/A 45 F/63 5550 3.3 BMAH Aldo and cortisol R/3.5 46 M/38 3185 3.4 APA Aldo R/1.3 47 M/72 5220 3.1 APA Aldo and cortisol R/1.0 48 M/38 2005 3.3 BMAH Aldo and cortisol L/10.0 49 M/53 5115 3.1 APA Aldo R/0.6 50 M/78 9025 1.9 APA Aldo L/2.0 Patient Number Sex/Age ARR (N < 550) (pmol/L:ng/mL/h) Serum K+ (mmol/L) Pathological Diagnosis Secretion Side (R/L)/Size (cm) 1 F/40 4030 3.4 APA Aldo L/1.7 2 M/38 3180 3.6 APA Aldo R/0.5 3 F/55 192 4.6 BMAH Aldo L/1.4 4 M/51 4425 3.2 BMAH Aldo R/1.1 5 F/52 1552 3.3 APA Aldo L/0.8 6 M/67 7795 3.9 BMAH Aldo R/1.5 7 F/66 N/A APA Aldo and cortisol R/1.2 8 M/42 1126 2.9 APA Aldo R/1.2 9 F/52 339 (pmol/L:pg/mL) 2.9 APA Aldo R/2.0 10 M/45 2163 2.9 APA Aldo L/0.8 11 M/47 7255 3.2 APA Aldo L/1.0 12 M/50 6500 2.8 BMAH Aldo L/1.3 13 F/38 8387 2.1 APA Aldo and cortisol R/1.3 14 F/32 5780 3.3 BMAH Aldo L/1.7 15 F/49 3145 3.2 BMAH Aldo and cortisol R/2.2 16 F/52 7260 3.3 APA Aldo and cortisol L/2.2 17 M/53 4043 4.5 BMAH Aldo R/1.2 18 M/53 320 3.2 APA Aldo and cortisol L/1.4 19 F/60 2828 3.4 BMAH Aldo and cortisol R/4.0 20 F/33 6460 3.3 APA Aldo L/1.8 21 F/34 3868 2.9 APA Aldo L/2.3 22 F/48 291 2.8 ACC and APA Aldo and cortisol D/2.0 23 M/72 1035 3.3 APA Aldo L/1.2 24 F/61 4063 3.9 APA Aldo L/1.0 25 M/54 2285 3.4 APA Aldo L/0.8 26 M/35 4570 3.9 APA Aldo L/1.5 27 F/71 5440 3.2 APA Aldo L/1.6 28 F/61 91 3.6 Oncocytoma Aldo and cortisol R/10.5 29 M/60 650 4.3 APA Aldo R/1.2 30 M/55 3289 3.4 APA Aldo L/0.9 31 F/54 134 4.5 BMAH Aldo and cortisol L/2.3 32 F/55 792 2.8 APA Aldo L/1.7 33 M/64 4322 4.3 APA Aldo L/0.9 34 F/69 18,341 3.7 APA Aldo R/3.0 35 M/51 9040 4.3 APA Aldo L/1.9 36 M/57 11,090 3.3 APA Aldo L/1.0 37 F/38 1060 3.1 APA Aldo L/1.3 38 F/59 4905 1.9 APA Aldo L/1.5 39 M/56 984 4.3 APA Aldo R/2.5 40 F/38 15,367 3.3 APA Aldo R/1.6 41 M/64 2513 3.1 APA Aldo L/0.5 42 F/43 297 3.4 IHA and cortisol-secreting adenoma Aldo and cortisol L/3.0 43 F/56 1180 3.2 APA Aldo L/0.8 44 F/28 2680 3.4 BMAH Aldo L/N/A 45 F/63 5550 3.3 BMAH Aldo and cortisol R/3.5 46 M/38 3185 3.4 APA Aldo R/1.3 47 M/72 5220 3.1 APA Aldo and cortisol R/1.0 48 M/38 2005 3.3 BMAH Aldo and cortisol L/10.0 49 M/53 5115 3.1 APA Aldo R/0.6 50 M/78 9025 1.9 APA Aldo L/2.0 Abbreviations: Aldo, aldosterone; F, female; L, left; M, male; N/A, not available; R, right. View Large Table 1. Clinical and Pathological Characteristics of Patients Patient Number Sex/Age ARR (N < 550) (pmol/L:ng/mL/h) Serum K+ (mmol/L) Pathological Diagnosis Secretion Side (R/L)/Size (cm) 1 F/40 4030 3.4 APA Aldo L/1.7 2 M/38 3180 3.6 APA Aldo R/0.5 3 F/55 192 4.6 BMAH Aldo L/1.4 4 M/51 4425 3.2 BMAH Aldo R/1.1 5 F/52 1552 3.3 APA Aldo L/0.8 6 M/67 7795 3.9 BMAH Aldo R/1.5 7 F/66 N/A APA Aldo and cortisol R/1.2 8 M/42 1126 2.9 APA Aldo R/1.2 9 F/52 339 (pmol/L:pg/mL) 2.9 APA Aldo R/2.0 10 M/45 2163 2.9 APA Aldo L/0.8 11 M/47 7255 3.2 APA Aldo L/1.0 12 M/50 6500 2.8 BMAH Aldo L/1.3 13 F/38 8387 2.1 APA Aldo and cortisol R/1.3 14 F/32 5780 3.3 BMAH Aldo L/1.7 15 F/49 3145 3.2 BMAH Aldo and cortisol R/2.2 16 F/52 7260 3.3 APA Aldo and cortisol L/2.2 17 M/53 4043 4.5 BMAH Aldo R/1.2 18 M/53 320 3.2 APA Aldo and cortisol L/1.4 19 F/60 2828 3.4 BMAH Aldo and cortisol R/4.0 20 F/33 6460 3.3 APA Aldo L/1.8 21 F/34 3868 2.9 APA Aldo L/2.3 22 F/48 291 2.8 ACC and APA Aldo and cortisol D/2.0 23 M/72 1035 3.3 APA Aldo L/1.2 24 F/61 4063 3.9 APA Aldo L/1.0 25 M/54 2285 3.4 APA Aldo L/0.8 26 M/35 4570 3.9 APA Aldo L/1.5 27 F/71 5440 3.2 APA Aldo L/1.6 28 F/61 91 3.6 Oncocytoma Aldo and cortisol R/10.5 29 M/60 650 4.3 APA Aldo R/1.2 30 M/55 3289 3.4 APA Aldo L/0.9 31 F/54 134 4.5 BMAH Aldo and cortisol L/2.3 32 F/55 792 2.8 APA Aldo L/1.7 33 M/64 4322 4.3 APA Aldo L/0.9 34 F/69 18,341 3.7 APA Aldo R/3.0 35 M/51 9040 4.3 APA Aldo L/1.9 36 M/57 11,090 3.3 APA Aldo L/1.0 37 F/38 1060 3.1 APA Aldo L/1.3 38 F/59 4905 1.9 APA Aldo L/1.5 39 M/56 984 4.3 APA Aldo R/2.5 40 F/38 15,367 3.3 APA Aldo R/1.6 41 M/64 2513 3.1 APA Aldo L/0.5 42 F/43 297 3.4 IHA and cortisol-secreting adenoma Aldo and cortisol L/3.0 43 F/56 1180 3.2 APA Aldo L/0.8 44 F/28 2680 3.4 BMAH Aldo L/N/A 45 F/63 5550 3.3 BMAH Aldo and cortisol R/3.5 46 M/38 3185 3.4 APA Aldo R/1.3 47 M/72 5220 3.1 APA Aldo and cortisol R/1.0 48 M/38 2005 3.3 BMAH Aldo and cortisol L/10.0 49 M/53 5115 3.1 APA Aldo R/0.6 50 M/78 9025 1.9 APA Aldo L/2.0 Patient Number Sex/Age ARR (N < 550) (pmol/L:ng/mL/h) Serum K+ (mmol/L) Pathological Diagnosis Secretion Side (R/L)/Size (cm) 1 F/40 4030 3.4 APA Aldo L/1.7 2 M/38 3180 3.6 APA Aldo R/0.5 3 F/55 192 4.6 BMAH Aldo L/1.4 4 M/51 4425 3.2 BMAH Aldo R/1.1 5 F/52 1552 3.3 APA Aldo L/0.8 6 M/67 7795 3.9 BMAH Aldo R/1.5 7 F/66 N/A APA Aldo and cortisol R/1.2 8 M/42 1126 2.9 APA Aldo R/1.2 9 F/52 339 (pmol/L:pg/mL) 2.9 APA Aldo R/2.0 10 M/45 2163 2.9 APA Aldo L/0.8 11 M/47 7255 3.2 APA Aldo L/1.0 12 M/50 6500 2.8 BMAH Aldo L/1.3 13 F/38 8387 2.1 APA Aldo and cortisol R/1.3 14 F/32 5780 3.3 BMAH Aldo L/1.7 15 F/49 3145 3.2 BMAH Aldo and cortisol R/2.2 16 F/52 7260 3.3 APA Aldo and cortisol L/2.2 17 M/53 4043 4.5 BMAH Aldo R/1.2 18 M/53 320 3.2 APA Aldo and cortisol L/1.4 19 F/60 2828 3.4 BMAH Aldo and cortisol R/4.0 20 F/33 6460 3.3 APA Aldo L/1.8 21 F/34 3868 2.9 APA Aldo L/2.3 22 F/48 291 2.8 ACC and APA Aldo and cortisol D/2.0 23 M/72 1035 3.3 APA Aldo L/1.2 24 F/61 4063 3.9 APA Aldo L/1.0 25 M/54 2285 3.4 APA Aldo L/0.8 26 M/35 4570 3.9 APA Aldo L/1.5 27 F/71 5440 3.2 APA Aldo L/1.6 28 F/61 91 3.6 Oncocytoma Aldo and cortisol R/10.5 29 M/60 650 4.3 APA Aldo R/1.2 30 M/55 3289 3.4 APA Aldo L/0.9 31 F/54 134 4.5 BMAH Aldo and cortisol L/2.3 32 F/55 792 2.8 APA Aldo L/1.7 33 M/64 4322 4.3 APA Aldo L/0.9 34 F/69 18,341 3.7 APA Aldo R/3.0 35 M/51 9040 4.3 APA Aldo L/1.9 36 M/57 11,090 3.3 APA Aldo L/1.0 37 F/38 1060 3.1 APA Aldo L/1.3 38 F/59 4905 1.9 APA Aldo L/1.5 39 M/56 984 4.3 APA Aldo R/2.5 40 F/38 15,367 3.3 APA Aldo R/1.6 41 M/64 2513 3.1 APA Aldo L/0.5 42 F/43 297 3.4 IHA and cortisol-secreting adenoma Aldo and cortisol L/3.0 43 F/56 1180 3.2 APA Aldo L/0.8 44 F/28 2680 3.4 BMAH Aldo L/N/A 45 F/63 5550 3.3 BMAH Aldo and cortisol R/3.5 46 M/38 3185 3.4 APA Aldo R/1.3 47 M/72 5220 3.1 APA Aldo and cortisol R/1.0 48 M/38 2005 3.3 BMAH Aldo and cortisol L/10.0 49 M/53 5115 3.1 APA Aldo R/0.6 50 M/78 9025 1.9 APA Aldo L/2.0 Abbreviations: Aldo, aldosterone; F, female; L, left; M, male; N/A, not available; R, right. View Large Pathological diagnosis was 35 APA (72%), 12 bilateral macronodular adrenal hyperplasia (BMAH) (24%), 1 bilateral hyperplasia (IHA) with a concomitant cortisol-secreting adenoma (2%), 1 oncocytoma (2%), and 1 adrenocortical carcinoma (ACC) with a concomitant APA (2%). Following adrenalectomy, follow-up for resolution of hypertension and/or hypokalemia was available in 45 cases (90%). Two cases showed resolution of the hypertension alone (4%), 14 cases had resolution of both the hypertension and the hypokalemia (31%), and 20 cases improved hypertension with resolution of hypokalemia (44%). Three cases persisted with hypertension alone (7%) and six cases with hypertension and hypokalemia (13%). Aldosterone response to stimulation with GnRH in vivo The in vivo stimulation with GnRH was performed in 23 patients, and a positive response (>50% renin-independent increase of aldosterone) was identified in seven patients (30.4%) (6 APAs and 1 BMAH), a partial response (>25% to 49% increase of aldosterone) in four patients (17.4%) (3 APAs and 1 IHA), and no response in 12 patients (52.2%) (7 APAs and 5 BMAHs) (Table 2). The in vivo stimulation with LH was performed in seven patients with GnRH response in aldosterone and showed a positive response (>50% renin-independent increase of aldosterone) in four patients (4 APAs) and no response in three patients (2 APAs and 1 IHA) (Table 2). Table 2. Responses to In Vivo Stimulation Test GnRH and/or LH and Genetic Analysis Patient Number Sex/Age Pathological Diagnosis Secretion GnRH (% of Aldosterone Elevation) LH (% of Aldosterone Elevation) CTNNB1 (Exon 3) GNAS (Exons 8 and 9) KCNJ5 (Exon 2) CACNA1D (Exons 6, 8A, 8B, 14, 16, 23, 27, and 33) ATP1A1 (Exons 4 and 8) ATP2B3 (Exon 8) 2 M/38 APA Aldo No response (–11%) N/A N/A N/A N/A N/A N/A N/A 3 F/55 BMAH Aldo No response (11%) N/A WT WT a WT WT WT 6 M/67 BMAH Aldo No response (–45%) N/A WT WT a WT WT WT 25 M/54 APA Aldo No response (–20%) N/A WT WT a WT c.311T>G, p.Leu104Argb WT 29 M/60 APA Aldo No response (–13%) N/A WT WT a WT WT WT 30 M/55 APA Aldo No response (18%) N/A WT WT c.837G>A, p.Glu279a,c c.776T>A, p.Val259Aspb WT WT 31 F/54 BMAH Aldo and cortisol No response (19%) N/A WT WT a WT WT WT 39 M/56 APA Aldo No response (3%) N/A WT WT a WT WT WT 40 F/38 APA Aldo No response (19%) No response (6%) WT WT c.451G>C, p.Gly151Arga,b WT WT WT 44 F/28 BMAH Aldo No response (18%) N/A WT WT a WT WT c.1224G>,
p.Pro408c 47 M/72 APA Aldo and cortisol No response (0%) N/A WT WT a WT WT WT 48 M/38 BMAH Aldo and cortisol No response (8%) N/A c.81A>G, p.Gln27c WT a WT WT WT 5 F/52 APA Aldo Partial response (47%) Positive response (100%) WT WT a WT WT WT 9 F/52 APA Aldo Partial response (42%) N/A WT WT a WT WT WT 26 M/35 APA Aldo Partial response (28%) N/A WT WT c.121C>T, p.Arg41Cysa,d c.451G>C, p.Gly151Argb WT WT WT 42 F/43 IHA Aldo and cortisol Partial response (42%) No response (N/A) WT c.601C>T, p.Arg201Cysb a WT WT WT 18 M/53 APA Aldo and cortisol Positive response (200%) Positive response (214%) WT WT a WT WT WT 24 F/61 APA Aldo Positive response (114%) N/A WT WT a WT WT WT 41 M/64 APA Aldo Positive response (118%) No response (9%) WT WT a WT WT WT 45 F/63 BMAH Aldo and cortisol Positive response (92%) N/A N/A N/A N/A N/A N/A N/A 46 M/38 APA Aldo Positive response (66%) Positive response (80%) WT WT a WT WT WT 49 M/53 APA Aldo Positive response (85%) N/A N/A N/A N/A N/A N/A N/A 50 M/78 APA Aldo Positive response (119%) Positive response (168%) WT WT a WT WT WT 21 F/34 APA Aldo N/A Positive response (65%) WT WT c.451G>C, p.Gly151Arga,b WT WT WT Patient Number Sex/Age Pathological Diagnosis Secretion GnRH (% of Aldosterone Elevation) LH (% of Aldosterone Elevation) CTNNB1 (Exon 3) GNAS (Exons 8 and 9) KCNJ5 (Exon 2) CACNA1D (Exons 6, 8A, 8B, 14, 16, 23, 27, and 33) ATP1A1 (Exons 4 and 8) ATP2B3 (Exon 8) 2 M/38 APA Aldo No response (–11%) N/A N/A N/A N/A N/A N/A N/A 3 F/55 BMAH Aldo No response (11%) N/A WT WT a WT WT WT 6 M/67 BMAH Aldo No response (–45%) N/A WT WT a WT WT WT 25 M/54 APA Aldo No response (–20%) N/A WT WT a WT c.311T>G, p.Leu104Argb WT 29 M/60 APA Aldo No response (–13%) N/A WT WT a WT WT WT 30 M/55 APA Aldo No response (18%) N/A WT WT c.837G>A, p.Glu279a,c c.776T>A, p.Val259Aspb WT WT 31 F/54 BMAH Aldo and cortisol No response (19%) N/A WT WT a WT WT WT 39 M/56 APA Aldo No response (3%) N/A WT WT a WT WT WT 40 F/38 APA Aldo No response (19%) No response (6%) WT WT c.451G>C, p.Gly151Arga,b WT WT WT 44 F/28 BMAH Aldo No response (18%) N/A WT WT a WT WT c.1224G>,
p.Pro408c 47 M/72 APA Aldo and cortisol No response (0%) N/A WT WT a WT WT WT 48 M/38 BMAH Aldo and cortisol No response (8%) N/A c.81A>G, p.Gln27c WT a WT WT WT 5 F/52 APA Aldo Partial response (47%) Positive response (100%) WT WT a WT WT WT 9 F/52 APA Aldo Partial response (42%) N/A WT WT a WT WT WT 26 M/35 APA Aldo Partial response (28%) N/A WT WT c.121C>T, p.Arg41Cysa,d c.451G>C, p.Gly151Argb WT WT WT 42 F/43 IHA Aldo and cortisol Partial response (42%) No response (N/A) WT c.601C>T, p.Arg201Cysb a WT WT WT 18 M/53 APA Aldo and cortisol Positive response (200%) Positive response (214%) WT WT a WT WT WT 24 F/61 APA Aldo Positive response (114%) N/A WT WT a WT WT WT 41 M/64 APA Aldo Positive response (118%) No response (9%) WT WT a WT WT WT 45 F/63 BMAH Aldo and cortisol Positive response (92%) N/A N/A N/A N/A N/A N/A N/A 46 M/38 APA Aldo Positive response (66%) Positive response (80%) WT WT a WT WT WT 49 M/53 APA Aldo Positive response (85%) N/A N/A N/A N/A N/A N/A N/A 50 M/78 APA Aldo Positive response (119%) Positive response (168%) WT WT a WT WT WT 21 F/34 APA Aldo N/A Positive response (65%) WT WT c.451G>C, p.Gly151Arga,b WT WT WT Positive response: elevation of aldosterone, >50%. Partial response: elevation of aldosterone, 25% to 50%. No response: elevation of aldosterone, <25%. Abbreviations: Aldo, aldosterone; F, female; M, male; N/A, not available; WT, wild-type. a Polymorphisms in KCNJ5 (c.171T>C, p.Ser57; c.810T>G, p.Leu270; and c.834T>C, p.His278). b Disease causing (pathogenic/likely pathogenic). c Polymorphism. d VUS. View Large Table 2. Responses to In Vivo Stimulation Test GnRH and/or LH and Genetic Analysis Patient Number Sex/Age Pathological Diagnosis Secretion GnRH (% of Aldosterone Elevation) LH (% of Aldosterone Elevation) CTNNB1 (Exon 3) GNAS (Exons 8 and 9) KCNJ5 (Exon 2) CACNA1D (Exons 6, 8A, 8B, 14, 16, 23, 27, and 33) ATP1A1 (Exons 4 and 8) ATP2B3 (Exon 8) 2 M/38 APA Aldo No response (–11%) N/A N/A N/A N/A N/A N/A N/A 3 F/55 BMAH Aldo No response (11%) N/A WT WT a WT WT WT 6 M/67 BMAH Aldo No response (–45%) N/A WT WT a WT WT WT 25 M/54 APA Aldo No response (–20%) N/A WT WT a WT c.311T>G, p.Leu104Argb WT 29 M/60 APA Aldo No response (–13%) N/A WT WT a WT WT WT 30 M/55 APA Aldo No response (18%) N/A WT WT c.837G>A, p.Glu279a,c c.776T>A, p.Val259Aspb WT WT 31 F/54 BMAH Aldo and cortisol No response (19%) N/A WT WT a WT WT WT 39 M/56 APA Aldo No response (3%) N/A WT WT a WT WT WT 40 F/38 APA Aldo No response (19%) No response (6%) WT WT c.451G>C, p.Gly151Arga,b WT WT WT 44 F/28 BMAH Aldo No response (18%) N/A WT WT a WT WT c.1224G>,
p.Pro408c 47 M/72 APA Aldo and cortisol No response (0%) N/A WT WT a WT WT WT 48 M/38 BMAH Aldo and cortisol No response (8%) N/A c.81A>G, p.Gln27c WT a WT WT WT 5 F/52 APA Aldo Partial response (47%) Positive response (100%) WT WT a WT WT WT 9 F/52 APA Aldo Partial response (42%) N/A WT WT a WT WT WT 26 M/35 APA Aldo Partial response (28%) N/A WT WT c.121C>T, p.Arg41Cysa,d c.451G>C, p.Gly151Argb WT WT WT 42 F/43 IHA Aldo and cortisol Partial response (42%) No response (N/A) WT c.601C>T, p.Arg201Cysb a WT WT WT 18 M/53 APA Aldo and cortisol Positive response (200%) Positive response (214%) WT WT a WT WT WT 24 F/61 APA Aldo Positive response (114%) N/A WT WT a WT WT WT 41 M/64 APA Aldo Positive response (118%) No response (9%) WT WT a WT WT WT 45 F/63 BMAH Aldo and cortisol Positive response (92%) N/A N/A N/A N/A N/A N/A N/A 46 M/38 APA Aldo Positive response (66%) Positive response (80%) WT WT a WT WT WT 49 M/53 APA Aldo Positive response (85%) N/A N/A N/A N/A N/A N/A N/A 50 M/78 APA Aldo Positive response (119%) Positive response (168%) WT WT a WT WT WT 21 F/34 APA Aldo N/A Positive response (65%) WT WT c.451G>C, p.Gly151Arga,b WT WT WT Patient Number Sex/Age Pathological Diagnosis Secretion GnRH (% of Aldosterone Elevation) LH (% of Aldosterone Elevation) CTNNB1 (Exon 3) GNAS (Exons 8 and 9) KCNJ5 (Exon 2) CACNA1D (Exons 6, 8A, 8B, 14, 16, 23, 27, and 33) ATP1A1 (Exons 4 and 8) ATP2B3 (Exon 8) 2 M/38 APA Aldo No response (–11%) N/A N/A N/A N/A N/A N/A N/A 3 F/55 BMAH Aldo No response (11%) N/A WT WT a WT WT WT 6 M/67 BMAH Aldo No response (–45%) N/A WT WT a WT WT WT 25 M/54 APA Aldo No response (–20%) N/A WT WT a WT c.311T>G, p.Leu104Argb WT 29 M/60 APA Aldo No response (–13%) N/A WT WT a WT WT WT 30 M/55 APA Aldo No response (18%) N/A WT WT c.837G>A, p.Glu279a,c c.776T>A, p.Val259Aspb WT WT 31 F/54 BMAH Aldo and cortisol No response (19%) N/A WT WT a WT WT WT 39 M/56 APA Aldo No response (3%) N/A WT WT a WT WT WT 40 F/38 APA Aldo No response (19%) No response (6%) WT WT c.451G>C, p.Gly151Arga,b WT WT WT 44 F/28 BMAH Aldo No response (18%) N/A WT WT a WT WT c.1224G>,
p.Pro408c 47 M/72 APA Aldo and cortisol No response (0%) N/A WT WT a WT WT WT 48 M/38 BMAH Aldo and cortisol No response (8%) N/A c.81A>G, p.Gln27c WT a WT WT WT 5 F/52 APA Aldo Partial response (47%) Positive response (100%) WT WT a WT WT WT 9 F/52 APA Aldo Partial response (42%) N/A WT WT a WT WT WT 26 M/35 APA Aldo Partial response (28%) N/A WT WT c.121C>T, p.Arg41Cysa,d c.451G>C, p.Gly151Argb WT WT WT 42 F/43 IHA Aldo and cortisol Partial response (42%) No response (N/A) WT c.601C>T, p.Arg201Cysb a WT WT WT 18 M/53 APA Aldo and cortisol Positive response (200%) Positive response (214%) WT WT a WT WT WT 24 F/61 APA Aldo Positive response (114%) N/A WT WT a WT WT WT 41 M/64 APA Aldo Positive response (118%) No response (9%) WT WT a WT WT WT 45 F/63 BMAH Aldo and cortisol Positive response (92%) N/A N/A N/A N/A N/A N/A N/A 46 M/38 APA Aldo Positive response (66%) Positive response (80%) WT WT a WT WT WT 49 M/53 APA Aldo Positive response (85%) N/A N/A N/A N/A N/A N/A N/A 50 M/78 APA Aldo Positive response (119%) Positive response (168%) WT WT a WT WT WT 21 F/34 APA Aldo N/A Positive response (65%) WT WT c.451G>C, p.Gly151Arga,b WT WT WT Positive response: elevation of aldosterone, >50%. Partial response: elevation of aldosterone, 25% to 50%. No response: elevation of aldosterone, <25%. Abbreviations: Aldo, aldosterone; F, female; M, male; N/A, not available; WT, wild-type. a Polymorphisms in KCNJ5 (c.171T>C, p.Ser57; c.810T>G, p.Leu270; and c.834T>C, p.His278). b Disease causing (pathogenic/likely pathogenic). c Polymorphism. d VUS. View Large Genetic analysis in the cohort of patients evaluated for aldosterone response to GnRH stimulation Among the 23 patients who underwent the in vivo stimulation with GnRH, we found no CTNNB1 mutations in the 11 patients with positive or partial response in aldosterone to GnRH. We found a silent CTNNB1 mutation, c.81A>G, p.Gln27, in a patient with aldosterone-secreting BMAH (patient 48) with no in vivo response to the GnRH. The c.601C>T, p.Arg201Cys GNAS mutation was found in the cortisol-secreting adenoma of a patient with concomitant IHA (patient 42), but no GNAS mutations were found in aldosterone-secreting tissues. This patient presented a partial response in aldosterone to GnRH and no response to LH stimulation. The somatic disease-causing KCNJ5 (c.451G>C, p.Gly151Arg) mutation was found in three patients with APA: One showed no response (patient 40), and one showed a positive response to LH stimulation, but was not tested with GnRH (patient 21). The third patient had a partial response (patient 26) of aldosterone to GnRH, and in addition to this disease-causing KCNJ5 somatic mutation, we identified in his leukocytes the germline KCNJ5 c.121C>T, p.Arg41Cys genetic alteration, which is a variant of uncertain significance (VUS). A ATP1A1 mutation (c.311T>G, p.Leu104Arg) (patient 25) and a CACNA1D mutation (c.776T>A, p.Val259Asp) (patient 30) were found in two patients (two APAs) with no response to in vivo stimulation with GnRH. Details of the responses to stimulation tests and various genetic alterations found are presented in Table 2 (Fig. 1; Supplemental Fig. 1). Figure 1. View largeDownload slide Sanger sequencing chromatograms showing somatic mutations p.Ser45Pro (CTNNB1), p.Arg201Cys (GNAS), p.Leu104Arg (ATP1A1), p.Gly151Arg (KCNJ5), p.Leu168Arg (KCNJ5), p.Val259Asp (CACNA1D), and p.Val1151Phe (CACNA1D). When the leukocyte DNA was not available, the adjacent normal tissue DNA was used. Figure 1. View largeDownload slide Sanger sequencing chromatograms showing somatic mutations p.Ser45Pro (CTNNB1), p.Arg201Cys (GNAS), p.Leu104Arg (ATP1A1), p.Gly151Arg (KCNJ5), p.Leu168Arg (KCNJ5), p.Val259Asp (CACNA1D), and p.Val1151Phe (CACNA1D). When the leukocyte DNA was not available, the adjacent normal tissue DNA was used. Genetic analysis in the cohort of patients not tested in vivo with GnRH stimulation Among the 26 patients who did not undergo the in vivo stimulation test with GnRH or LH, we found two pathogenic CTNNB1 mutations (c.133T>C, p.Ser45Pro): in a patient with BMAH cosecreting aldosterone and cortisol (patient 19) and 26 995 del 271 bp in one patient with APA (patient 34) (24). The somatic disease-causing KCNJ5 (c.451G>C, p.Gly151Arg) mutation was found in four patients (three APAs and one BMAH), and the somatic disease-causing KCNJ5 (c.451G>A, p.Gly151Arg) mutation was found in six patients (five APAs and one BMAH). The KCNJ5 mutation (c.503T>G, p.Leu168Arg) was found in one BMAH and two APAs. One CACNA1D mutation, c.3451G>T, p.Val1151Phe, was found in one patient with APA. No GNAS, ATP1A1, or ATP2B3 mutations were found. All details of genetic alterations found in the cohort of patients not tested in vivo with GnRH stimulation are presented in Table 3. Table 3. Genetic Analysis in Adrenal Tissues of Patients Without In Vivo Aberrant Receptor Stimulation Tests Patient Number Sex/Age Pathological Diagnosis Secretion CTNNB1 (Exon 3) GNAS (Exons 8 and 9) KCNJ5 (Exon 2) CACNA1D (Exons 6, 8A, 8B, 14, 16, 23, 27, and 33) ATP1A1 (Exons 4 and 8) ATP2B3 (Exon 8) 1 F/40 APA Aldo WT WT c.451G>A, p.Gly151Arga,b WT WT WT 4 M/51 BMAH Aldo WT WT a WT WT WT 7 F/66 APA Aldo and cortisol WT WT c.451G>C, p.Gly151Arga,b WT WT WT 8 M/42 APA Aldo WT WT c.224G>A, p.Ser75Asna,c WT WT WT 10 M/45 APA Aldo WT WT a WT WT WT 11 M/47 APA Aldo WT WT a WT WT WT 12 M/50 BMAH Aldo WT WT c.451G>C, p.Gly151Arga,b WT WT WT 13 F/38 APA Aldo and cortisol WT WT a WT WT WT 14 F/32 BMAH Aldo WT WT c.503T>G, p.Leu168Arga,b WT WT WT 15 F/49 BMAH Aldo and cortisol WT WT c.451G>A,p.Gly151Arga,b WT WT WT 16 F/52 APA Aldo and cortisol WT WT c.451G>A, p.Gly151Arga,b WT WT WT 17 M/53 BMAH Aldo WT WT a WT WT WT 19 F/60 BMAH Aldo and cortisol c.133T>C, p.Ser45Prob WT a WT WT WT 20 F/33 APA Aldo WT WT c.503T>G, p.Leu168Argb WT WT WT 22 F/48 ACC and APA Aldo and cortisol WT WT c.451G>A, p.Gly151Arga,b WT WT WT 23 M/72 APA Aldo WT WT a WT WT WT 27 F/71 APA Aldo WT WT c.503T>G, p.Leu168Arga,b WT WT WT 28 F/61 Oncocytoma Aldo and cortisol WT WT a WT WT WT 32 F/55 APA Aldo WT WT c.451G>C, p.Gly151Arga,b WT WT WT 33 M/64 APA Aldo WT WT a WT WT WT 34 F/69 APA Aldo 26 995 del 271 bpb p.Ala5_Ala80del WT a WT WT WT 35 M/51 APA Aldo WT WT c.451G>C, p.Gly151Arga,b WT WT WT 36 M/57 APA Aldo WT WT a WT WT WT 37 F/38 APA Aldo WT WT a c.3451G>T, p.Val1151Pheb WT WT 38 F/59 APA Aldo WT WT c.451G>A, p.Gly151Arga,b WT WT WT 43 F/56 APA Aldo WT WT c.451G>A, p.Gly151Arga,b WT WT WT Patient Number Sex/Age Pathological Diagnosis Secretion CTNNB1 (Exon 3) GNAS (Exons 8 and 9) KCNJ5 (Exon 2) CACNA1D (Exons 6, 8A, 8B, 14, 16, 23, 27, and 33) ATP1A1 (Exons 4 and 8) ATP2B3 (Exon 8) 1 F/40 APA Aldo WT WT c.451G>A, p.Gly151Arga,b WT WT WT 4 M/51 BMAH Aldo WT WT a WT WT WT 7 F/66 APA Aldo and cortisol WT WT c.451G>C, p.Gly151Arga,b WT WT WT 8 M/42 APA Aldo WT WT c.224G>A, p.Ser75Asna,c WT WT WT 10 M/45 APA Aldo WT WT a WT WT WT 11 M/47 APA Aldo WT WT a WT WT WT 12 M/50 BMAH Aldo WT WT c.451G>C, p.Gly151Arga,b WT WT WT 13 F/38 APA Aldo and cortisol WT WT a WT WT WT 14 F/32 BMAH Aldo WT WT c.503T>G, p.Leu168Arga,b WT WT WT 15 F/49 BMAH Aldo and cortisol WT WT c.451G>A,p.Gly151Arga,b WT WT WT 16 F/52 APA Aldo and cortisol WT WT c.451G>A, p.Gly151Arga,b WT WT WT 17 M/53 BMAH Aldo WT WT a WT WT WT 19 F/60 BMAH Aldo and cortisol c.133T>C, p.Ser45Prob WT a WT WT WT 20 F/33 APA Aldo WT WT c.503T>G, p.Leu168Argb WT WT WT 22 F/48 ACC and APA Aldo and cortisol WT WT c.451G>A, p.Gly151Arga,b WT WT WT 23 M/72 APA Aldo WT WT a WT WT WT 27 F/71 APA Aldo WT WT c.503T>G, p.Leu168Arga,b WT WT WT 28 F/61 Oncocytoma Aldo and cortisol WT WT a WT WT WT 32 F/55 APA Aldo WT WT c.451G>C, p.Gly151Arga,b WT WT WT 33 M/64 APA Aldo WT WT a WT WT WT 34 F/69 APA Aldo 26 995 del 271 bpb p.Ala5_Ala80del WT a WT WT WT 35 M/51 APA Aldo WT WT c.451G>C, p.Gly151Arga,b WT WT WT 36 M/57 APA Aldo WT WT a WT WT WT 37 F/38 APA Aldo WT WT a c.3451G>T, p.Val1151Pheb WT WT 38 F/59 APA Aldo WT WT c.451G>A, p.Gly151Arga,b WT WT WT 43 F/56 APA Aldo WT WT c.451G>A, p.Gly151Arga,b WT WT WT Abbreviations: Aldo, aldosterone; F, female; M, male; N/A, not available; WT, wild-type. a Polymorphisms in KCNJ5 (c.171T>C, p.Ser57; c.810T>G, p.Leu270; and c.834T>C, p.His278). b Disease causing (pathogenic/likely pathogenic). c VUS. View Large Table 3. Genetic Analysis in Adrenal Tissues of Patients Without In Vivo Aberrant Receptor Stimulation Tests Patient Number Sex/Age Pathological Diagnosis Secretion CTNNB1 (Exon 3) GNAS (Exons 8 and 9) KCNJ5 (Exon 2) CACNA1D (Exons 6, 8A, 8B, 14, 16, 23, 27, and 33) ATP1A1 (Exons 4 and 8) ATP2B3 (Exon 8) 1 F/40 APA Aldo WT WT c.451G>A, p.Gly151Arga,b WT WT WT 4 M/51 BMAH Aldo WT WT a WT WT WT 7 F/66 APA Aldo and cortisol WT WT c.451G>C, p.Gly151Arga,b WT WT WT 8 M/42 APA Aldo WT WT c.224G>A, p.Ser75Asna,c WT WT WT 10 M/45 APA Aldo WT WT a WT WT WT 11 M/47 APA Aldo WT WT a WT WT WT 12 M/50 BMAH Aldo WT WT c.451G>C, p.Gly151Arga,b WT WT WT 13 F/38 APA Aldo and cortisol WT WT a WT WT WT 14 F/32 BMAH Aldo WT WT c.503T>G, p.Leu168Arga,b WT WT WT 15 F/49 BMAH Aldo and cortisol WT WT c.451G>A,p.Gly151Arga,b WT WT WT 16 F/52 APA Aldo and cortisol WT WT c.451G>A, p.Gly151Arga,b WT WT WT 17 M/53 BMAH Aldo WT WT a WT WT WT 19 F/60 BMAH Aldo and cortisol c.133T>C, p.Ser45Prob WT a WT WT WT 20 F/33 APA Aldo WT WT c.503T>G, p.Leu168Argb WT WT WT 22 F/48 ACC and APA Aldo and cortisol WT WT c.451G>A, p.Gly151Arga,b WT WT WT 23 M/72 APA Aldo WT WT a WT WT WT 27 F/71 APA Aldo WT WT c.503T>G, p.Leu168Arga,b WT WT WT 28 F/61 Oncocytoma Aldo and cortisol WT WT a WT WT WT 32 F/55 APA Aldo WT WT c.451G>C, p.Gly151Arga,b WT WT WT 33 M/64 APA Aldo WT WT a WT WT WT 34 F/69 APA Aldo 26 995 del 271 bpb p.Ala5_Ala80del WT a WT WT WT 35 M/51 APA Aldo WT WT c.451G>C, p.Gly151Arga,b WT WT WT 36 M/57 APA Aldo WT WT a WT WT WT 37 F/38 APA Aldo WT WT a c.3451G>T, p.Val1151Pheb WT WT 38 F/59 APA Aldo WT WT c.451G>A, p.Gly151Arga,b WT WT WT 43 F/56 APA Aldo WT WT c.451G>A, p.Gly151Arga,b WT WT WT Patient Number Sex/Age Pathological Diagnosis Secretion CTNNB1 (Exon 3) GNAS (Exons 8 and 9) KCNJ5 (Exon 2) CACNA1D (Exons 6, 8A, 8B, 14, 16, 23, 27, and 33) ATP1A1 (Exons 4 and 8) ATP2B3 (Exon 8) 1 F/40 APA Aldo WT WT c.451G>A, p.Gly151Arga,b WT WT WT 4 M/51 BMAH Aldo WT WT a WT WT WT 7 F/66 APA Aldo and cortisol WT WT c.451G>C, p.Gly151Arga,b WT WT WT 8 M/42 APA Aldo WT WT c.224G>A, p.Ser75Asna,c WT WT WT 10 M/45 APA Aldo WT WT a WT WT WT 11 M/47 APA Aldo WT WT a WT WT WT 12 M/50 BMAH Aldo WT WT c.451G>C, p.Gly151Arga,b WT WT WT 13 F/38 APA Aldo and cortisol WT WT a WT WT WT 14 F/32 BMAH Aldo WT WT c.503T>G, p.Leu168Arga,b WT WT WT 15 F/49 BMAH Aldo and cortisol WT WT c.451G>A,p.Gly151Arga,b WT WT WT 16 F/52 APA Aldo and cortisol WT WT c.451G>A, p.Gly151Arga,b WT WT WT 17 M/53 BMAH Aldo WT WT a WT WT WT 19 F/60 BMAH Aldo and cortisol c.133T>C, p.Ser45Prob WT a WT WT WT 20 F/33 APA Aldo WT WT c.503T>G, p.Leu168Argb WT WT WT 22 F/48 ACC and APA Aldo and cortisol WT WT c.451G>A, p.Gly151Arga,b WT WT WT 23 M/72 APA Aldo WT WT a WT WT WT 27 F/71 APA Aldo WT WT c.503T>G, p.Leu168Arga,b WT WT WT 28 F/61 Oncocytoma Aldo and cortisol WT WT a WT WT WT 32 F/55 APA Aldo WT WT c.451G>C, p.Gly151Arga,b WT WT WT 33 M/64 APA Aldo WT WT a WT WT WT 34 F/69 APA Aldo 26 995 del 271 bpb p.Ala5_Ala80del WT a WT WT WT 35 M/51 APA Aldo WT WT c.451G>C, p.Gly151Arga,b WT WT WT 36 M/57 APA Aldo WT WT a WT WT WT 37 F/38 APA Aldo WT WT a c.3451G>T, p.Val1151Pheb WT WT 38 F/59 APA Aldo WT WT c.451G>A, p.Gly151Arga,b WT WT WT 43 F/56 APA Aldo WT WT c.451G>A, p.Gly151Arga,b WT WT WT Abbreviations: Aldo, aldosterone; F, female; M, male; N/A, not available; WT, wild-type. a Polymorphisms in KCNJ5 (c.171T>C, p.Ser57; c.810T>G, p.Leu270; and c.834T>C, p.His278). b Disease causing (pathogenic/likely pathogenic). c VUS. View Large Mutational analysis of the entire cohort of patients with PA Overall, two disease-causing mutations of the CTNNB1 gene were found among the 47 patients that were studied with PA (prevalence of 4.3%). One of 47 (2%) samples harbored a GNAS mutations (c.601C>T, p.Arg201Cys) in a cortisol-secreting adenoma with concomitant IHA. Somatic pathogenic/likely pathogenic KCNJ5 mutations were identified in 16/47 (34%) tissues studied, including c.451G>A, p.Gly151Arg (n = 6), c.451G>C, p.Gly151Arg (n = 7) (11 APAs and 2 BMAHs), and c.503T>G, p.Leu168Arg (n = 3) (2 APAs and 1 BMAH). In addition, two KCNJ5 germline VUSs, c.224G>A, p.Ser75Asn and c.121C>T, p.Arg41Cys, were identified in DNA leukocytes of two patients with APA. Two pathogenic CACNA1D mutations (4%), c.776T>A, p.Val259Asp in exon 6, and c.3451G>T, p.Val1151Phe in exon 27, were found in two APA samples. Moreover, a somatic pathogenic ATP1A1 mutation (2%) (c.311T>G, p.Leu104Arg) was identified in one APA. RT-PCR expression of GNRHR and LHCGR and Western blotting/immunohistochemical analysis of β-catenin protein As shown in Fig. 2, there was no significant statistical difference between mRNA expression of GNRHR and LHCGR in patients with positive, partial, or no response in aldosterone to GnRH in vivo. mRNA LHCGR expression was increased in two out of three patients with in vivo response to LH in aldosterone (Fig. 2), but not in patients with no response to LH. Western blotting analysis showed variable expression of β-catenin protein in patients with positive, partial, or no response to GnRH (Fig. 3). Immunohistochemical analysis of the adrenocortical tissue of patient 19 harboring a pathogenic CTNNB1 mutation showed diffuse, membranous staining for β-catenin (3+) and nuclear staining in isolated cells supporting activation of the Wnt/β-catenin signaling in this BMAH cosecreting cortisol and aldosterone. Figure 2. View largeDownload slide Relative mRNA expression of (A) GNRHR and (B) LHCGR in adrenal tissues from patients with PA: three with no response, three with partial response, and six with positive aberrant response of aldosterone to in vivo stimulation with GnRH. (C) Relative mRNA expression of LHCGR in a subgroup of the patients who were also tested for aldosterone response to LH administration: two with no response and three with positive response to LH. The relative quantification was performed by the 2–ΔΔCT method, using a pool of normal adrenal glands as calibrator and GADD45A as endogenous control gene. Each point represents a patient. GADD45A, human α growth arrest and DNA damage inducible. Figure 2. View largeDownload slide Relative mRNA expression of (A) GNRHR and (B) LHCGR in adrenal tissues from patients with PA: three with no response, three with partial response, and six with positive aberrant response of aldosterone to in vivo stimulation with GnRH. (C) Relative mRNA expression of LHCGR in a subgroup of the patients who were also tested for aldosterone response to LH administration: two with no response and three with positive response to LH. The relative quantification was performed by the 2–ΔΔCT method, using a pool of normal adrenal glands as calibrator and GADD45A as endogenous control gene. Each point represents a patient. GADD45A, human α growth arrest and DNA damage inducible. Figure 3. View largeDownload slide Western blot analysis of β-catenin protein in adrenal tissues from patients with PA: two with no response, two with partial response, and thre with positive response of aldosterone to in vivo stimulation with GnRH. Each point represents a patient. The values under the bands show results of densitometric analysis. They were normalized to actin and to control sample. The control (Ctl) sample is a pooled homogenate from all of the samples. Figure 3. View largeDownload slide Western blot analysis of β-catenin protein in adrenal tissues from patients with PA: two with no response, two with partial response, and thre with positive response of aldosterone to in vivo stimulation with GnRH. Each point represents a patient. The values under the bands show results of densitometric analysis. They were normalized to actin and to control sample. The control (Ctl) sample is a pooled homogenate from all of the samples. Discussion Our study combined the search for genetic alterations in six genes implicated in PA with in vivo aberrant regulation of aldosterone by GnRH and/or LH. Close to 50% of the 23 patients with PA increased their aldosterone following stimulation with GnRH or LH (four of seven responding to LH); previously Zwermann et al. (25) found a 25% response in 12 patients studied and Albiger et al. (18) found a 83% response in 12 patients (3 positive and 7 partial responses). The in vivo stimulation with GnRH was previously evaluated by 2 independent groups in 13 healthy subjects, and no increase in aldosterone was found in any (18, 25). We found two somatic disease-causing CTNNB1 mutations in two patients (one BMAH and one APA) who were not tested in vivo for a GnRH response. However, no CTNNB1 mutations were found among the 11 patients who had a positive or partial aberrant response of aldosterone to GnRH. Our findings are different from a previous report where CTNNB1 mutations were found in APA of two pregnant women and one postmenopausal woman, and a link was suggested between the CTNNB1 mutations and the aberrant expression of LHCGR and GNRHR (19). Another group described four women with CTNNB1 mutations in their APA, without association with pregnancy in any of the cases (26). In addition, a third group reported no CTNNB1 mutations among 26 APAs (27). In our cohort, the CTNNB1 mutations were found in two women of 60 and 69 years of age. Our sample size is too small to conclude to higher prevalence of CTNNB1 mutations in woman, neither at older age, as previously reported by others (16, 17). Unfortunately, the duration of hypertension before the surgery was unknown, but following surgery, complete remission occurred in two patients with a CTNNB1 mutation. A study reported a higher proportion of residual hypertension after adrenalectomy in patients with a CTNNB1 mutation, but this cannot be evaluated in our small sample size (17). Although, the activation of the WNT/β-catenin pathway is a major feature in APA, CTNNB1 mutations were identified in only 4% of our cohort (15). This is similar to two previous cohorts where a prevalence of 5.1% of somatic CTNNB1 mutations among 198 APAs and 3.7% among 209 APAs was found (16, 17). Thus, the prevalence of CTNNB1 mutations in APA remains much lower than nonsecreting or cortisol-secreting adrenocortical tumors (24, 28). The molecular mechanisms associated with CTNNB1 mutations in PA are still unclear. Recently, it was suggested that CTNNB1 mutations may be more related to tumorigenesis than to excessive aldosterone production (17, 29). In our cohort, CTNNB1 mutations occurred mutually exclusively to KCNJ5, GNAS, ATP1A1, ATP2B3, or CACNAD1 mutations. This is concordant with findings in two other cohorts of patients with APA (16, 17). We found an overall prevalence of 34% of pathogenic/likely pathogenic KCNJ5 mutations among our total cohort of 47 patients, which is similar to previous reports (5–8). Among those patients, one presented a partial response to stimulation with GnRH (patient 26) and another (patient 21) had a positive response to stimulation with LH. One study evaluated the expression of GNRHR and LHCGR in APA with (n = 13) and without (n = 9) KCNJ5 mutations (30). They found that GNRHR and LHCGR were highly expressed among APAs with no KCNJ5 mutations. Unfortunately, due to tissue availability, we studied a small number of adrenal tissues for GNRHR and LHCGR mRNA expression that showed variable expression not clearly correlated to the in vivo response to GnRH. In addition, according to the sites of the biopsy, results might be variables because of the heterogeneity of the adrenal tissues. We found a prevalence of 4% of CACNA1D mutations and 2% of ATP1A1 mutations. One of each was tested for GnRH response, and in both cases, there was no response to the in vivo GnRH stimulation. To our knowledge, no previous study has specifically looked at the expression of GNRHR in APA with CACNA1D, ATP1A1, or ATP2B3 mutations. There were strengths and limitations to our study. To our knowledge, this is the first study to compare the prevalence of six mutations implicated in PA with aberrant aldosterone stimulation by GnRH and/or LH in vivo. Unfortunately, not all patients were able to participate in the in vivo aberrant receptor screening protocol before their surgery. We found a similar prevalence of CTNNB1 mutations in our cohort as in previously published series. Further studies are needed to clarify the link between CTNNB1 mutations and development of PA. As previously suggested, it may be more related to tumorigenesis rather than to aldosterone production itself. Finally, CTNNB1 mutations do not lead to all in vivo GnRH positive or partial response in PA, as none of our patients with aberrant aldosterone regulation by GnRH or LH carried a somatic CTNNB1 mutation in their adrenocortical tissues. Abbreviations: Abbreviations: ACC adrenocortical carcinoma APA aldosterone-producing adenoma ARR aldosterone-to-renin ratio BMAH bilateral macronodular adrenal hyperplasia GnRH gonadotropin-releasing hormone GNRHR gonadotropin-releasing hormone receptor IHA bilateral hyperplasia LH luteinizing hormone LHCGR luteinizing hormone/choriogonadotropin receptor PA primary aldosteronism VUS variant of uncertain significance Acknowledgments Financial Support: This work was supported in part by a salary grant from Fonds de Recherche du Québec-Santé (to I.B.) and the Canadian Institutes of Health and Research (to I.B. and A.L.). Disclosure Summary: The authors have nothing to disclose. References 1. 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Journal of Clinical Endocrinology and MetabolismOxford University Press

Published: Aug 1, 2018

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