KCNJ5 mutations in aldosterone- and cortisol-co-secreting adrenal adenomas

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1 Endocrine Journal, 59 (8),735- Rapid Communication Advance Publication doi:.57/endocrj. EJ-47 KCNJ5 mutations in aldosterone- and cortisol-co-secreting adrenal adenomas Masanobu Yamada*, Yasuyo Nakajima*, Ryo Taguchi*, Takashi Okamura, Sumiyasu Ishii, Takuya Tomaru, Atsushi Ozawa, Nobuyuki Shibusawa, Satoshi Yoshino, Akiko Toki, Emi Ishida, Koshi Hashimoto, Tetsurou Satoh and Masatomo Mori Department of Medicine and Molecular Science, Gunma University Graduate School of Medicine, Maebashi 37-85, Japan Abstract. Adrenal aldosterone-producing adenomas (APA) are rarely associated with the clear co-secretion of cortisol. Somatic mutations of the potassium channel KCNJ5 gene, with the hotspots G5R and L68R, have been recently identified in patients with APA. However, whether APAs that secrete cortisol have these mutations remains unclear. We examined three patients with APAs showing clear autonomous secretion of cortisol who possessed a mg dexamethasone suppression test (DST) with a failure of the serum cortisol level to drop below 3. µg/dl, a morning plasma ACTH level of less than pg/ml, and suppressed accumulation in the intact adrenal on 3 I- adosterol scintigraphy, or postoperative adrenal insufficiency. Laparoscopic adrenectomy revealed all tumors to be golden yellow, and histological examination confirmed them to be adrenocortical adenomas. All these patients required replacement therapy with hydrocortisone after surgery. Sequencing demonstrated that of three cases showed a mutation of the KCNJ5 gene, one with c.45g>a, p.g5r and one with c.53t>g, p.l68r. Furthermore, the mrna levels of steroidogenic enzymes including CYPB, CYPB, HSD3B, CYP7A, CYPA and KCNJ5 in the 3 cases did not differ from those in 8 pure APAs not showing any of the above conditions for autonomous cortisol secretion. In addition, all 8 pure APAs harbored mutations of the KCNJ5 gene. These findings suggested that at least some aldosterone- and cortisol-co-secreting adrenal tumors have mutations of the KCNJ5 gene, suggesting the origin to be APA, and pure APAs may show a high incidence of KCNJ5 mutations. Key words: KCNJ5, Aldosterone-producing adenoma, Adrenocortical adenomas, Co-secretion of cortisol Primary aldosteronism (PA) has been reported to be more common than expected and to affect at least 6~% of hypertensive patients. PA is commonly caused by an adrenal adenoma of the adrenal cortex (aldosterone-producing adenoma, APA), by unilateral or bilateral adrenal hyperplasia (idiopathic hyperaldosteronism, IHA), or in rare cases, by inherited glucocorticoid-remediable aldosteronism (GRA) []. In Western countries, about one third of patients with PA have APA [-5], in contrast, most Japanese with PA (~8%) have APA [6-9]. Another characteristic feature of Japanese patients is that the APA sometimes associated with mild autonomous cortisol secre- Submitted Jul., ; Accepted Jul. 9, as EJ-47 Released online in J-STAGE as advance publication Aug. 3, Correspondence to: Masanobu Yamada, M.D., Ph.D., Department of Medicine and Molecular Science, Gunma University Graduate School of Medicine, Showa-machi, Maebashi, Gunma 37-85, Japan. myamada@gunma-u.ac.jp *These three authors equally contributed. tion. In fact, among our recent 76 patients with PA, about 3 % showed either a mg dexamethasone suppression test (DST) with a failure of the serum cortisol level to suppress to less than 3. µg/dl or a midnight cortisol level of more than 7.4 µg/dl [9]. Importantly, a relatively high percentage of patients with this association had a history of cardiovascular events [9]. Although the mechanisms underlying the tumorigenesis of APA had been largely unknown, Choi et al. successfully identified somatic mutations (G5R and L68R) in the gene encoding the potassium channel KCNJ5 (also known as Kir 3.4 or GIRK4) in 8 of (about 36%) patients with APA []. The two somatic mutations (G5R and L68R) are located in and near the selectivity filter of KCNJ5, and have been reported to cause increased sodium conductance and membrane depolarization resulting in the activation of voltagegated Ca + channels. Then, rising intracellular Ca + levels increase aldosterone production and promotes increased proliferation resulting in an adenoma []. The Japan Endocrine Society

2 Yamada et al. We and others have recently confirmed the same two mutations (G5R and L68R) of the KCNJ5 gene in APAs in Japan, Australia, the United Kingdom, and Europe. We found mutations of the KCNJ5 gene in 5 of 3 (about 65%) APAs in Japan, and Azizan et al. reported a lower frequency, 38% in Australia and 44% in the United Kingdom [, ]. Boulkroun et al. examined the largest number, 38 cases of APA, and found that 34% patients had mutations in Europe [3]. In the present study, since the concept of preclinical (or subclinical) Cushing s syndrome is not universally accepted [4], we selected cases who possessed all following conditions; mg dexamethasone suppression test (DST) with a failure of the serum cortisol level to drop below 3. µg/dl, a morning plasma ACTH level of less than pg/ml, and suppressed accumulation in the intact adrenal on 3 I-adosterol scintigraphy, or postoperative adrenal insufficiency. We then examined whether these APAs associated with the clear co-secretion of cortisol have mutations of the KCNJ5 gene, and measured mrna levels for several steroidogenic enzymes and compared them to those in pure APAs not showing any above parameters for autonomous cortisol secretion. Subjects and Methods Subjects Each subject provided written informed consent, and the study was approved by the ethics committee on human research of Gunma University. The diagnosis of PA was performed as reported previously [, 9, 5]. RNA extraction and detection of mutations of KCNJ5 cdna by direct sequencing All specimens of APA and some normal parts of the adrenal cortex were frozen in liquid nitrogen immediately after removal during the operation. Total RNA was prepared, and cdna was reverse-transcribed and sequenced with specific primers sets for the entire KCNJ5 cdna as reported previously []. Expression of KCNJ5 and steroidogenic enzyme mrna levels determined by Real-time PCR To measure the level of KCNJ5 and steroidogenic enzyme mrna in each adenoma,.5 µl of cdna was subjected to real-time PCR as reported [6, 7]. All reactions were performed in triplicate using TaqMan probes and an Applied Biosystems 75 sequence detection system. The TaqMan probes for KCNJ5 (Hs68476_m), GAPDH (Hs _ g), cholesterol side-chain cleavage enzyme, CYPA (Hs67984_m), CYPB (Hs59644_m), CYPB (Hs59773_m), 3β-hydroxysteroid dehydrogenase (HSD3B) (Hs653_m), and 7αhydroxylase (CYP7A) (Hs436_m) were from Applied Biosystems. The level of each mrna relative to that of GAPDH was calculated using a standard curve, and relative quantification was performed as described in ABI User Bulletin #. All experiments were repeated at least twice. The level of mrna expression for each patient was evaluated with the median value for the normal adrenal cortex set as.. Hormone measurement Plasma aldosterone levels were measured in most cases with the RIA SPAC-S Aldosterone kit TFB; plasma renin activity, with the RIA Renin IRMA KIT Daiichi TFB; plasma cortisol, with the RIA Cortisol kit TFB TFB; and ACTH, with ECLIA Eclusys ACTH by Roche Diagnostics. Statistical analysis All results are expressed as the median or mean ± SD for continuous variables. Group comparisons were performed with an ANOVA and Student s t test for normally distributed data, or the Wilcoxon rank-sum test or Mann-Whitney test for non-normally distributed data for continuous variables. All tests for significance and resulting P values were two-sided, with a level of significance of 5%. Statistical analyses were performed using JMP 5.. (SAS Institute Inc. Cary, NC, USA). Results Case reports Case. A 5-year-old woman had hypertension and hypokalemia with. meq/l and a symptom of Cushing s syndrome, moon face. Her plasma aldosterone concentration (PAC) was 33. ng/dl, plasma renin activity (PRA),.6 ng/ml/hr; morning serum cortisol level, 3. µg/dl; serum ACTH level, < 5 pg/ ml. She showed suppressed accumulation in the intact adrenal on scintigraphy. Her midnight cortisol level at 3: pm was.3 µg/dl. After a mg DST, serum cortisol level was 3 µg/dl. A CT scan demonstrated a tumor 4.5 cm in diameter in the left adrenal gland. The maximum standardized uptake value (maxsuv) on F-8 fluorodeoxyglucose-positron emission tomography (FDG-PET) was 4.8.

3 KCNJ5 in APA with co-secretion of cortisol 3 Table Clinical features of 3 patients with APA and clear co-secretion of cortisol. Pt Age (yr) Gender PAC (ng/dl) PRA (ng/ml/h) F8 (μg/dl) F3 (μg/dl) ACTH (pg/dl) mgdst (μg/dl) Scintigraphy SBP DBP Serum (mmhg) (mmhg) K (meq/l) Spironolactone (mg) K supplement (g) Tumor size (mm) KCNJ5 mutation 5 F < G5RG/A 48 F WT 3 53 F L68R Pt indicates the number of the patient; Age, the age when APA was diagnosed; F, female. F8 indicates the morning cortisol level around 8: AM after a 3-minute bed rest. Plasma aldosterone (PAC), plasma renin activity (PRA), and plasma ACTH levels were also measured at the same time. The midnight cortisol level (F3) around : PM was measured at least 3 days after the admission. mg DST, serum cortisol level after a mg dexamethasone suppression test. SBP and DBP, systolic and diastolic blood pressure. The doses of Spironolactone and potassium (K) supplement taken before surgery are indicated. All the patients were given spironolactone for at least one week before the surgery. Tumor size is the maximum diameter of the tumor observed in the CT study. Scintigraphy, suppressed accumulation in the intact adrenal gland. G5RG/A represents a heterozygous mutation of c.45g>a (p.g5r) and L68R for c.53t>g (p.l68r). WT represents wild-type alleles. Pt. 3 showed insufficiency of adrenocortical function after the surgery. Case. A 48-year-old woman had hypertension and hypokalemia with 3.4 meq/l, but did not have any symptoms of Cushing s syndrome. Her PAC was 3. ng/dl, PRA,.4 ng/ml/hr, serum cortisol level, 9.9 µg/ dl, and serum ACTH level, 6.8 pg/ml. She showed suppressed accumulation in the intact adrenal on scintigraphy. Her midnight cortisol level was 5. µg/dl. After a mg DST, her serum cortisol level was 7. µg/ dl. A CT scan demonstrated a tumor 3. cm in diameter in the left adrenal gland. This patient corresponds to patient in Table in our previous study []. Case 3. A 53-year-old woman had hypertension and hypokalemia with.3 meq/l, but did not have symptoms of Cushing s syndrome. She had had cerebral breeding six months before admission. Her PAC was 39.3 ng/dl, PRA,. ng/ml/hr, morning serum cortisol level, 6.6 µg/dl, and serum ACTH level, 34 pg/ ml. She showed reduced accumulation in the intact adrenal on scintigraphy. Her midnight cortisol level was.4 µg/dl. After a mg DST, her serum cortisol level was 4. µg/dl. A CT scan demonstrated a tumor 3. cm in diameter in the left adrenal gland. MRI did not show any tumor in the pituitary. A CRH test showed a rapid increase in ACTH but no elevation of cortisol in response to the elevated ACTH. A rapid ACTH test showed normal response of cortisol after 3 min. She showed insufficiency of adrenocortical function after surgery, including hypotension, hyponatremia and a reduced level of consciousness. The tumor was confirmed to be an adrenocortical adenoma and there was no staining with an ACTH antibody. Therefore, her ACTH before the surgery might have been inactive possibly due to the recent cerebral bleeding. Furthermore, histological examination confirmed that all three APAs (Cases ~3) were isolated adenomas and there was no additional micro-cortisol producing adenoma. Characteristics of patients with APA (pure APA) not showing any criteria indicating autonomous cortisol secretion We had 8 operated samples of APAs showing no parameters of autonomous cortisol secretion including the mg DST with a failure to suppress the serum cortisol level to less than 3. µg/dl, a morning plasma ACTH level to less than pg/ml, and the accumulation of intact adrenal on scintigraphy or post-operated adrenocortical insufficiency. We also excluded cases who had missed any value for these parameters. We defined these APAs as pure APAs in the present study. Six of 8 patients correspond to Pt., 3, 9,, and 3 in Table in our previous study []. The additional cases were a 5-year-old male, and a 36-yearold female, and their PAC, PRA and serum potassium levels were 94.4 and 54. ng/dl,. and. ng/ml/h, and.6 and.5 meq/l, respectively. The mean age of these 8 patients was 4 ± 3 years, and 38 percent were male. The mean PAC was 35.4 ± 7. ng/dl (7.6~94.4), PRA, mean.4 ±.3 ng/ml/hr (.~.9) and the mean serum potassium level,.7 ±.6 meq/l (.~3.5). The maximum tumor diameter measured by CT was.5 ±.4 cm (.~.3). Mutations of KCNJ5 in APAs showing autonomous cortisol secretion and pure APAs Analysis of the KCNJ5 cdna in APAs demonstrated that patient had somatic mutations of the KCNJ5

4 4 Yamada et al. A B CYPB mrna/gapdh 6 4 CYPB mrna/gapdh C D CYP7A mrna/gapdh HSD3B mrna/gapdh 3 E F CYPA mrna/gapdh.5.5 Fig. Expression of steroidogenic enzymes and KCNJ5 mrna levels in APAs with co-secretion of cortisol and pure APAs Black dots indicate the values for APAs with co-secretion of cortisol and gray dots, pure APAs. Pt indicates the corresponding number of the patient in Table. KCNJ5 mrna/gapdh cortisol, to be increased in APAs showing co-secretion of cortisol. Therefore, we examined the expression of CYPB and other steroidogenic enzymes mrna including CYPB, CYP7A, HSD3B, CYPA as well as KCNJ5. In Fig., black circles indicate values of the patients with APAs showing co-secretion of cortisol and indicate the corresponding patient number in Table, while gray circles represents those of pure APAs. For all steroidogenic enzyme and KCNJ5 mrna levels, the value for the normal adrenal gland adjust to the APA was set as.. As shown in Fig. A, as expected, levels of CYPB mrna in most tumors including 3 APAs with co-secretion of cortisol were higher than or at least similar to those in normal adregene;p.g5r, c.45g>a, and patient 3 had the mutation p.l68r, c.53t>g. These mutations were heterozygous. In contrast, no mutation of the KCNJ5 gene was observed in the APA of patient No.. It is important to note that all 8 pure APAs showed mutations of the KCNJ5 gene; 4 with c.45g>a, p.g5r, with c.53t>g, p.l68r and with c.45g>c, p.g5r. Expression of steroidogenic enzymes and KCNJ5 mrna in patients with APAs showing autonomous cortisol secretion and pure APAs We expected a steroidogenic enzyme such as CYPB, which is responsible for the production of

5 KCNJ5 in APA with co-secretion of cortisol 5 nal gland. Unexpectedly, the CYPB mrna levels in APAs with co-secretion of cortisol did not differ from those in pure APAs (Fig. B). Furthermore, neither CYP7A, HSD3B nor CYPA mrna levels differed from those in pure APAs (Fig. C, D and E). In addition, no significant difference in KCNJ5 mrna levels was observed between the APAs with co-secretion of cortisol and the pure APAs (Fig. F). Discussion In the present study, of 3 APAs showing clear co-secretion of cortisol had a mutation of the KCNJ5 gene. Boulkroun et al. recently examined 6 patients with cortisol-producing adenomas (CPA) who showed typical symptoms of Cushing s syndrome and found no mutations of the KCNJ5 gene [3]. We also examined 6 cases of CPA (submitted), but they did not have the mutation. Therefore, although the number of CPAs examined was small, considering the high prevalence (at least 3~%) of the mutation of the KCNJ5 gene in APA in several countries, the mutation may be highly specific to aldosterone-producing adenomas and familiar aldosteronism type III. Therefore, at least some of the APAs with co-secretion of cortisol appeared to have mutations of KCNJ5, suggesting them to be derived from APA, not from CPA. To confirm this, we performed qpcr for several steroidogenic enzymes, which may reflect their origin of the zone in the adrenal gland. However, the mrna levels of steroidogenic enzymes of 3 APAs with co-secretion of cortisol did not show any specific profile. Hiraishi et al. also reported that in a histopathological and immunohistochemical study of 3β-HSD and CYP7, CYPB mrna levels in APAs with subclinical Cushing s syndrome diagnosed with the guideline of a Research Committee for Adrenal Diseases supported by the Japanese Ministry of Health, Labor and Welfare did not show any difference from those in APAs [8]. Conversely, it is of interest that all 8 pure APAs harbored a KCNJ5 gene mutation, who did not show any parameter for autonomous cortisol secretion such as the mg DST with a failure of the serum cortisol level to drop to less than 3. µg/dl, a morning plasma ACTH level of less than pg/ml, and suppressed accumulation of intact adrenal in scintigraphy or postoperative adrenal insufficiency. In a previous study, we found about 65 % of 3 Japanese patients with APA to have a somatic mutation of the KCNJ5 gene []. Among these 3 patients, 6 had pure APAs as defined in the present study, and all of which had the mutation of the KCNJ5 gene. Monticone et al. recently reported a study of steroidogenic enzyme mrna levels in 47 APAs including 5 Japanese patients, and found all 5 cases to have a mutation of the KCNJ5 gene [9]. Although the details for these 5 cases were not reported, Japanese patients with typical APA may have a high prevalence of somatic mutations of the KCNJ5 gene. A lager scale study is required to reach a conclusion as to the precise prevalence of mutations of the KCNJ5 gene in Japanese with pure APAs. Among patients with adrenocortical adenomas, those with no typical symptoms of Cushing s syndrome (CS) such as moon face, buffalo hump etc. but showing the autonomous secretion of cortisol have been identified as having preclinical (or subclinical) Cushing s syndrome [, ]. Although the concept of preclinical (or subclinical) CS is not universally accepted [4], Spath et al. recently reported that we should consider an APA with co-secretion of cortisol if a patient has ) PA and an adenoma that is larger than.5 cm, ) cortisol that is non-suppressible with overnight low-dose dexamethasone, or 3) grossly elevated serum levels of hybrid steroids, such as 8-OH-F []. In fact, although we did not have a chance to measure serum hybrid steroids, all three cases had a tumor larger than.5 cm. These findings further support that the larger the APA, the higher the frequency of co-secretion of cortisol. The exact prevalence and clinical and pathological features of APA with co-secretion of cortisol have not been fully elucidated. Hogan et al. first reported an aldosterone- and cortisol-producing adenoma in 977 [3]. Subsequently, several papers have been published regarding the association of PA with the autonomous secretion of cortisol [, 4, 5]. Many such reports have come from Japan or Europe. Piaditis et al. have reported that PA with hypercortisolism was observed in.% of 83 adrenal incidentalomas [6]. In Japan, the prevalence was reported to be 5~3% [9, 8, 7]. Although the reason for the high prevalence of an association with autonomous cortisol secretion in APAs remains unclear, many cases have been described by Japanese investigators and this may have led to greater awareness in Japan []. Further study is required to investigate the regional difference in the prevalence of APA with co-secretion of cortisol and to establish a universal concept of subclinical Cushing s syndrome.

6 6 Yamada et al. Acknowledgements Intractable Disease (M. Y.). We thank all the medical and co-medical staff, and graduate students involved with the patients care. This study was supported in part by a Health and Labor Sciences Grant for Research on Measures for Disclosure Summary All authors have nothing to disclose. References. Funder JW, Carey RM, Fardella C, Gomez-Sanchez CE, Mantero F, Stowasser M, Young WF, Jr., Montori VM (8) Case detection, diagnosis, and treatment of patients with primary aldosteronism: An Endocrine Society clinical practice guideline. J Clin Endocrinol Metab 93: Rossi GP, Bernini G, Caliumi C, Desideri G, Fabris B, Ferri C, Ganzaroli C, Giacchetti G, Letizia C, Maccario M, Mallamaci F, Mannelli M, Mattarello MJ, Moretti A, Palumbo G, Parenti G, Porteri E, Semplicini A, Rizzoni D, Rossi E, Boscaro M, Pessina AC, Mantero F (6) A prospective study of the prevalence of primary aldosteronism in,5 hypertensive patients. J Am Coll Cardiol 48: Rossi GP, Seccia TM, Pessina AC (8) Primary aldosteronism - part I: prevalence, screening, and selection of cases for adrenal vein sampling. J Nephrol : Young WF (7) Primary aldosteronism: renaissance of a syndrome. Clin Endocrinol (Oxf) 66: Stowasser M, Gordon RD (3) Primary aldosteronism. Best Pract Res Clin Endocrinol Metab 7: Nishikawa T, Saito J, Omura M (7) Prevalence of primary aldosteronism: should we screen for primary aldosteronism before treating hypertensive patients with medication? Endocr J 54: Omura M, Saito J, Yamaguchi K, Kakuta Y, Nishikawa T (4) Prospective study on the prevalence of secondary hypertension among hypertensive patients visiting a general outpatient clinic in Japan. Hypertens Res 7: Omura M, Sasano H, Saito J, Yamaguchi K, Kakuta Y, Nishikawa T (6) Clinical characteristics of aldosterone-producing microadenoma, macroadenoma, and idiopathic hyperaldosteronism in 93 patients with primary aldosteronism. Hypertens Res 9: Nakajima Y, Yamada M, Taguchi R, Satoh T, Hashimoto K, Ozawa A, Shibusawa N, Okada S, Monden T, Mori M () Cardiovascular complications of patients with aldosteronism associated with autonomous cortisol secretion. J Clin Endocrinol Metab 96: Choi M, Scholl UI, Yue P, Bjorklund P, Zhao B, Nelson- Williams C, Ji W, Cho Y, Patel A, Men CJ, Lolis E, Wisgerhof MV, Geller DS, Mane S, Hellman P, Westin G, Akerstrom G, Wang W, Carling T, Lifton RP () K+ channel mutations in adrenal aldosterone-producing adenomas and hereditary hypertension. Science 33: Taguchi R, Yamada M, Nakajima Y, Satoh T, Hashimoto K, Shibusawa N, Ozawa A, Okada S, Rokutanda N, Takata D, Koibuchi Y, Horiguchi J, Oyama T, Takeyoshi I, Mori M () Expression and mutations of KCNJ5 mrna in Japanese patients with aldosterone-producing adenomas. J Clin Endocrinol Metab 97: Azizan EA, Murthy M, Stowasser M, Gordon R, Kowalski B, Xu S, Brown MJ, O Shaughnessy KM () Somatic mutations affecting the selectivity filter of KCNJ5 are frequent in large unselected collections of adrenal aldosteronomas. Hypertension 59: Boulkroun S, Beuschlein F, Rossi GP, Golib-Dzib JF, Fischer E, Amar L, Mulatero P, Samson-Couterie B, Hahner S, Quinkler M, Fallo F, Letizia C, Allolio B, Ceolotto G, Cicala MV, Lang K, Lefebvre H, Lenzini L, Maniero C, Monticone S, Perrocheau M, Pilon C, Plouin PF, Rayes N, Seccia TM, Veglio F, Williams TA, Zinnamosca L, Mantero F, Benecke A, Jeunemaitre X, Reincke M, Zennaro MC () Prevalence, clinical, and molecular correlates of KCNJ5 mutations in primary aldosteronism. Hypertension 59: Tsagarakis S, Vassiliadi D, Thalassinos N (6) Endogenous subclinical hypercortisolism: Diagnostic uncertainties and clinical implications. J Endocrinol Invest 9: Nishikawa T, Omura M, Satoh F, Shibata H, Takahashi K, Tamura N, Tanabe A () Guidelines for the diagnosis and treatment of primary aldosteronism -The Japan Endocrine Society 9. Endocr J 58: Ishida E, Yamada M, Horiguchi K, Taguchi R, Ozawa A, Shibusawa N, Hashimoto K, Satoh T, Yoshida S, Tanaka Y, Yokota M, Tosaka M, Hirato J, Yamada S, Yoshimoto Y, Mori M () Attenuated expression of menin and p7 (Kip) in an aggressive case of multiple endocrine neoplasia type (MEN) associated with an atypical prolactinoma and a malignant pancreatic endocrine tumor. Endocr J 58: Horiguchi K, Yamada M, Satoh T, Hashimoto K, Hirato J, Tosaka M, Yamada S, Mori M (9) Transcriptional activation of the mixed lineage leukemia-p7kip

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