KCNJ5 Mutations Research Articles

Somatic GNAQ, CTNNB1, and CACNA1C Mutations in Cat Aldosterone-Secreting Tumors.

Primary aldosteronism (PA) is a common cause of human hypertension. Somatic mutations in KCNJ5, CACNA1D, ATP1A1, and ATP2B3 are found in at least 80% of aldosterone-producing adenomas, which cause unilateral PA in humans. Somatic mutations have been identified infrequently in 7 other genes; few of these were known to play a role in aldosterone secretion before the discovery of their mutations. Interrogating somatic mutations in the domestic cat, in which spontaneous PA is also known to occur, might improve the understanding of normal adrenal gland physiology and the pathophysiology of PA. DNA and RNA extracted from tissue from 13 cats with unilateral aldosterone-secreting tumors, including 8 carcinomas and 5 adenomas, underwent whole genome sequencing, targeted Sanger sequencing, and RNA sequencing. Single-nucleotide substitution variants were filtered to select those with a predicted deleterious effect on protein function and a suspected role in aldosterone secretion. Probable functional somatic single-nucleotide polymorphisms (n=8) were found in 3 adenomas and 2 carcinomas. Mutations with predicted significant effects were identified in 2 genes also mutated in human PA; GNAQ and CTNNB1, and in a residue of CACNA1C analogous to a common CACNA1D mutation. In contrast to humans, CACNA1C expression was much greater than CACNA1D in both feline tumor and nontumor adrenal tissue. No mutations were identified in KCNJ5, CACNA1D, ATP1A1, or ATP2B3. Similar mutations were identified in cats to those found in humans. It is, therefore, likely that both species have shared underlying selection pressures for mutations that increase aldosterone secretion.

Diagnostic and therapeutic approaches to a case of pregnancy complicated by bilateral adrenocortical adenomas with primary aldosteronism and Cushing's syndrome.

Aldosterone/cortisol co-secreting adenomas (A/CPA) are a rare type of primary aldosteronism(PA), and cases of aldosterone/cortisol co-secreting adenomas during pregnancy are extremely rare, with no reported cases to date. The unique physiological state of pregnancy increases cortisol secretion through the hypothalamic-pituitary-adrenal (HPA) axis and leads to elevated levels of all components of the renin-angiotensin-aldosterone system (RAAS). This can cause overlapping symptoms with abnormal cortisol and aldosterone secretion, making diagnosis very challenging. This case involves a 29-year-old woman who developed hypercortisolism at 33 weeks of pregnancy. Despite receiving treatment for her symptoms and having a successful delivery, she continued to experience hypertension and hypokalaemia after giving birth. Eventually, she was diagnosed with ACTH-independent Cushing's syndrome and primary aldosteronism due to independent cortisol and aldosterone secretion from bilateral adrenal adenomas. Following a thorough diagnosis, classification, treatment, and follow-up, the patient achieved a clinical cure while preserving normal adrenal function. Further investigation revealed that both diseases were caused by KCNJ5 and PRKACA mutations found in the bilateral adrenal adenomas.

8294 Association between gene mutations and DNA methylation of CYP11B2 in aldosterone-producing adenoma

Abstract Disclosure: M. Kometani: None. R. Mizoguchi: None. M. Demura: None. K. Aiga: None. S. Konishi: None. D. Aono: None. S. Karashima: None. Y. Takeda: None. T. Yoneda: None. Context: Primary aldosteronism (PA) causes hypertension and cardiovascular complications. Recently, in aldosterone producing adenomas (APA), various genetic mutations have been identified. In addition, epigenetic mechanisms, such as DNA methylation, are also involved in excessive aldosterone production. Several studies have shown that aldosterone synthase (CYP11B2) is hypomethylated in APA. On the other hand, the relationship between specific genetic variants identified in APA and DNA methylation has not been fully clarified. Objective: To investigate the relationship between specific genetic mutations in APA and DNA methylation of CYP11B2. Methods: We analyzed 48 patients (female 23 cases) diagnosed with APA and treated between 2001 and 2022. First, Gene mutation analysis using next-generation sequencing was performed. KCNJ5, ATP1A1, ATP2B3, CACNA1D, and CACNA1H, which are known mutations in PA, and PRKACA, PRKA1A, GNAS, CTNNB1, and ARMC5, which are known mutations in Cushing’s syndrome, were evaluated. Next, DNA methylation analysis of the CYP11B2 promoter using pyrosequencing were performed using APA. We targeted three CpG regions near the transcription start site of CYP11B2. Results: The respective mean values for all patients were: age 52 years, systolic blood pressure 138 mmHg, diastolic blood pressure 87 mmHg, ARR 1,326, serum potassium level 3.4 mEq/L, and tumor diameter 15 mm. Genetic mutation analysis revealed KCNJ5 gene mutations in 33 of the 48 patients. Among the other cases, mutations in the CTNNB1 gene were found in three cases, mutations in the ATP1A1 gene in two cases, mutations in the CACNA1D gene in one case, and PRKACA gene mutations in one case. The remaining seven patients had no mutations in any of the analyzed genes. When DNA methylation of the promoter region of CYP11B2 was analyzed in APAs with and without mutations in KCNJ5, we found that in all three CpG regions, DNA methylation rate was significantly reduced in APAs with mutations in KCNJ5 gene (CpG1 22% vs. 30%, CpG2 42% vs. 73%, CpG3 47% vs. 75%, respectively). Conclusions: The most common genetic mutation in APA was the KCNJ5 mutation; APA with the KCNJ5 mutation had significantly lower methylation rates than CYP11B2. This suggests that genetic mutations in APA affect DNA methylation and result in hormone overproduction. Presentation: 6/3/2024

6966 A Rhythmic Life-Threatening Enigma Unveiled: KCNH2 Mutation-Associated Torsades de Points Unmasking Primary Hyperaldosteronism in Cardiological Realm

Abstract Disclosure: S. Dejprapasorn: None. N. Nopparatana: None. S. Soonthornpun: None. R. Leelawattana: None. N. Kietsiriroje: None. S. Limumpornpetch: None. T. Nilmoje: None. W. Lohawijarn: None. T. Wongsuttipakorn: None. P. Choochuen: None. P. Limumpornpetch: None. Introduction: Primary hyperaldosteronism (PA) is characterized by hypertension and hypokalemia. However, its ability to masquerade life-threatening Torsades de Pointes makes it an unusual and dismissed diagnosis, revealing the link between PA and serious arrythmia. Clinical Case A 30-year-old female experienced three episodes of palpitations, followed by syncope. Upon admission, her blood pressure was 147/87 mmHg. ECG showed prolonged QT intervals with potassium level was 2.6 mmol/L (3.5-5.0 mmol/L). Continuous cardiac monitoring captured Torsades de Pointes. Despite potassium replacement, long QT still persisted. Pathogenic variant, c.2592+1G>A, within KCNH2 was discovered. Aldosterone concentration showed 69.65 ng/dL (9.70-62.60 ng/dL) and plasma renin activity was 0.12 ng/mL/h (1.50-5.70 ng/mL/h). Aldosterone-to-renin ratio was 580. Aldosterone post 4-h saline infusion test was 31.71 ng/dL which were consistent with PA. Adrenal unenhanced CT scan identified bilateral adrenal nodules (right 1.3 cm, left 1.1 cm). After receiving a cardioverter-defibrillator and spironolactone, the patient has had no palpitations, and their potassium level has remained normal. Clinical lessons: PA rarely presents ventricular arrhythmia. KCNH2 mutation may link between long QT syndrome (LQTS) and PA arises. LQTS type 2 is attributable to KCNH2 mutations, impacting potassium channel subunits crucial for adjusting currents during sympathetic activation. This is essential for shortening the QT interval with increasing heart rate. KCNH2 mutations disrupt this process, prolonging the QT interval and heightening the risk of arrhythmias. Aldosterone synthesis relies on calcium signaling triggered by potassium currents. Adrenal cortex hosts approximately 90 potassium channels genes, including KCNJ5 and KCNH2. Mutations in gene can disrupt the calcium signaling cascade, leading to excessive aldosterone production. While KCNJ5 mutation is well-documented in PA, the potential correlation between KCNH2 mutations and a similar mechanism remains unexplored in clinical reports to date. This case highlights the importance of a thorough comprehension of genetic cause contributing to pathogenesis of PA with complex clinical presentations. Presentation: 6/1/2024

Double CYP11B1/CYP11B2 Immunohistochemistry and Detection of KCNJ5 Mutations in Primary Aldosteronism.

The search for somatic mutations in adrenals resected from patients with primary aldosteronism (PA) is performed by Sanger sequencing, often implemented with immunohistochemistry (IHC)-guidance focused on aldosterone-producing (CYP11B2-positive) areas. To investigate the impact of double IHC for CYP11B1 and CYP11B2 on Sanger and next-generation sequencing (NGS). We investigated 127 consecutive adrenal aldosterone-producing adenomas from consenting surgically cured PA patients using double IHC for CYP11B1 and CYP11B2, by Sanger sequencing and NGS. Double IHC for CYP11B2 and CYP11B1 revealed 3 distinct patterns: CYP11B2-positive adenoma (pattern 1), mixed CYP11B1/CYP11B2-positive adenoma (pattern 2), and adrenals with multiple small CYP11B2-positive nodules (pattern 3). Sanger sequencing allowed detection of KCNJ5 mutations in 44% of the adrenals; NGS revealed such mutations in 10% of those negative at Sanger and additional mutations in 61% of the cases. Importantly the rate of KCNJ5 mutations differed across patterns: 17.8% in pattern 1, 71.4% in pattern 2, and 10.7% in pattern 3 (χ2 = 22.492, P < .001). NGS allowed detection of mutations in many adrenals that tested negative at Sanger sequencing. Moreover, the different distribution of KCNJ5 mutations across IHC patterns indicates that IHC-guided sequencing protocols selecting CYP11B2-positive areas could furnish results that might not be representative of the entire mutational status of the excised adrenal, which is important at a time when KCNJ5 mutations are suggested to drive management of patients with aldosterone-producing adenomas.

The influence of cortisol co-secretion on clinical characteristics and postoperative outcomes in unilateral primary aldosteronism.

The prevalence of unilateral primary aldosteronism (UPA) with cortisol co-secretion varies geographically. To investigate the prevalence and clinical characteristics of UPA with cortisol co-secretion in a Chinese population. Retrospective cohort study. We recruited 580 patients with UPA who underwent cosyntropin stimulation test (CST) after the 1-mg dexamethasone suppression test (DST) and retrospectively analyzed the clinical characteristics and postoperative outcomes of UPA with and without cortisol co-secretion. UPA with cortisol co-secretion (1 mg DST>1.8 ug/dL) was identified in 65 of 580 (11.2%) patients. These patients were characterized by older age, longer duration of hypertension, higher concentration of plasma aldosterone and midnight cortisol, lower adrenocorticotropic hormone (ACTH) and dehydroepiandrosterone sulfate (DHEAS), larger tumor diameter, and more history of diabetes mellitus. Cortisol and aldosterone levels were higher and DHEAS level was lower in UPA with cortisol co-secretion at 0-120 min after CST. Among 342 UPA patients with KCNJ5 gene sequencing and follow-up results, the complete clinical success rate was lower in UPA with cortisol co-secretion (33.3% vs. 56.4%, P<0.05); the complete biochemical success rate and KCNJ5 mutation did not differ between the two groups. Age, tumor size, and ACTH were independent predictors of UPA with cortisol co-secretion. Sex, BMI, duration of hypertension, KCNJ5 mutation, and cortisol co-secretion were independent predictors for complete clinical success in UPA after surgery. UPA with cortisol co-secretion is not uncommon in China, but the clinical features were distinctly different from those without co-secretion. Cortisol co-secretion is an independent risk factor for incomplete clinical success after surgery in UPA.

Urine steroid metabolomics as a diagnostic tool in primary aldosteronism

Primary aldosteronism (PA) causes 5–10% of hypertension cases, but only a minority of patients are currently diagnosed and treated because of a complex, stepwise, and partly invasive workup. We tested the performance of urine steroid metabolomics, the computational analysis of 24-hour urine steroid metabolome data by machine learning, for the identification and subtyping of PA. Mass spectrometry-based multi-steroid profiling was used to quantify the excretion of 34 steroid metabolites in 24-hour urine samples from 158 adults with PA (88 with unilateral PA [UPA] due to aldosterone-producing adenomas [APAs]; 70 with bilateral PA [BPA]) and 65 sex- and age-matched healthy controls. All APAs were resected and underwent targeted gene sequencing to detect somatic mutations associated with UPA. Patients with PA had increased urinary metabolite excretion of mineralocorticoids, glucocorticoids, and glucocorticoid precursors. Urine steroid metabolomics identified patients with PA with high accuracy, both when applied to all 34 or only the three most discriminative steroid metabolites (average areas under the receiver-operating characteristics curve [AUCs-ROC] 0.95–0.97). Whilst machine learning was suboptimal in differentiating UPA from BPA (average AUCs-ROC 0.65–0.73), it readily identified APA cases harbouring somatic KCNJ5 mutations (average AUCs-ROC 0.79–85). These patients showed a distinctly increased urine excretion of the hybrid steroid 18-hydroxycortisol and its metabolite 18-oxo-tetrahydrocortisol, the latter identified by machine learning as by far the most discriminative steroid. In conclusion, urine steroid metabolomics is a non-invasive candidate test for the accurate identification of PA cases and KCNJ5-mutated APAs.

Single-Nucleus Analysis Reveals Tumor Heterogeneity of Aldosterone-Producing Adenoma.

Recent advances in omics techniques have allowed detailed genetic characterization of aldosterone-producing adenoma (APA). The pathogenesis of APA is characterized by tumorigenesis-associated aldosterone synthesis. The pathophysiological intricacies of APAs have not yet been elucidated at the level of individual cells. Therefore, a single-cell level analysis is speculated to be valuable in studying the differentiation process of APA. We conducted single-nucleus RNA sequencing of APAs with KCNJ5 mutation and nonfunctional adenomas obtained from 3 and 2 patients, respectively. The single-nucleus RNA sequencing revealed the intratumoral heterogeneity of APA and identified cell populations consisting of a shared cluster of nonfunctional adenoma and APA. In addition, we extracted 2 cell fates in APA and obtained a cell population specialized in aldosterone synthesis. Genes related to ribosomes and neurodegenerative diseases were upregulated in 1 of these fates, whereas those related to the regulation of glycolysis were upregulated in the other fate. Furthermore, the total RNA reads in the nucleus were higher in hormonally activated clusters, indicating a marked activation of transcription per cell. The single-nucleus RNA sequencing revealed intratumoral heterogeneity of APA with KCNJ5 mutation. The observation of 2 cell fates in KCNJ5-mutated APAs provides the postulation that a heterogeneous process of cellular differentiation was implicated in the pathophysiological mechanisms underlying APA tumors.

The W101C KCNJ5 Mutation Induces Slower Pacing by Constitutively Active GIRK Channels in hiPSC-Derived Cardiomyocytes.

Mutations in the KCNJ5 gene, encoding one of the major subunits of cardiac G-protein-gated inwardly rectifying K+ (GIRK) channels, have been recently linked to inherited forms of sinus node dysfunction. Here, the pathogenic mechanism of the W101C KCNJ5 mutation underlying sinus bradycardia in a patient-derived cellular disease model of sinus node dysfunction (SND) was investigated. A human-induced pluripotent stem cell (hiPSCs) line of a mutation carrier was generated, and CRISPR/Cas9-based gene targeting was used to correct the familial mutation as a control line. Both cell lines were further differentiated into cardiomyocytes (hiPSC-CMs) that robustly expressed GIRK channels which underly the acetylcholine-regulated K+ current (IK,ACh). hiPSC-CMs with the W101C KCNJ5 mutation (hiPSCW101C-CM) had a constitutively active IK,ACh under baseline conditions; the application of carbachol was able to increase IK,ACh, further indicating that not all available cardiac GIRK channels were open at baseline. Additionally, hiPSCW101C-CM had a more negative maximal diastolic potential (MDP) and a slower pacing frequency confirming the bradycardic phenotype. Of note, the blockade of the constitutively active GIRK channel with XAF-1407 rescued the phenotype. These results provide further mechanistic insights and may pave the way for the treatment of SND patients with GIRK channel dysfunction.

OR02-04 Peripheral Blood Steroid Profiling Discriminates Aldosterone-producing Adenoma Genotypes From Bilateral Primary Aldosteronism

Abstract Disclosure: A.F. Turcu: None. C. Lee: None. Z. Salman: None. H. Liu: None. A. Rabbah: None. S.M. Konzen: None. W.E. Rainey: None. T.J. Giordano: None. L. Zhao: None. A. Udager: None. Background: Primary aldosteronism (PA) is dichotomized into 2 major subtypes: unilaterally dominant PA, often caused by aldosterone-producing adenomas (APAs), and bilateral PA (BPA). Adrenal vein sampling (AVS)-guided unilateral adrenalectomy is often curative in patients with APA and offers marked cardiovascular and renal benefits. The diagnosis and subtyping of PA is a complex, multistep process, hindered by expertise and resource limitations. Objective: To develop steroid fingerprints that can distinguish genotype specific APAs from BPA in peripheral blood. Design And Methods: We enrolled 120 patients with PA who underwent AVS in a single tertiary referral center. Liquid-chromatography tandem mass spectrometry was used to simultaneously quantify 17 steroids in adrenal veins (AV) and periphery, both at baseline, and 10-20 min after cosyntropin stimulation. Aldosterone synthase (CYP11B2) immunohistochemistry-guided next generation sequencing was employed to identify aldosterone-driver mutations. Kruskal-Wallis tests were performed for comparison of continuous variables across multiple groups. Receiver operating characteristic (ROC) curves were plotted to discriminate APAs from BPA. Results: The 120 study participants included 75 patients with APA who underwent unilateral adrenalectomy and had documented aldosterone-driver mutations, and 45 patients with BPA. Of patients with APAs, 36 carried somatic mutations in CACNA1D, 24 in ATPase, and 15 in KCNJ5 genes, respectively. Patients with KCNJ5 mutations were younger and most were women. In the dominant AVs, the KCNJ5 group had marked elevations of 18-hydroxycortisol, 18-oxocortisol, aldosterone, and 11-deoxycorticosterone (DOC); conversely, the BB group had the lowest concentrations of mineralocorticoid and androgen precursors, despite comparable cortisol levels across all groups. Similar patterns were observed in peripheral blood. Linear discriminant analysis incorporating all steroids measured in baseline peripheral serum showed areas under the curve (AUC) of 0.98, 0.97, and 0.92 for distinguishing APAs with KCNJ5, ATPase, or CACNA1D mutations, respectively, from BPA. Conclusions: Single-assay steroid profiles measured in peripheral serum can distinguish genotype-specific APAs from BPA. Once validated, such tests might circumvent the need for AVS in large subset of PA patients. Presentation: Thursday, June 15, 2023

OR02-02 Pre-operative Blood Pressure Response To Aldosterone Antagonists And Urinary Hybrid Steroid Ratios Predict Clinical Outcomes In Unilateral Primary Aldosteronism For At Least 2 Years Post-adrenalectomy

Abstract Disclosure: X. Wu: None. E. Goodchild: None. R. Senanayake: None. W. Bashari: None. J. Salsbury: None. C.P. Cabrera: None. G. Argentesi: None. S.M. O’Toole: None. J. McFarlane: None. M. Matson: None. L. Parvanta: None. N. Hilliard: None. V. Kosmoliaptsis: None. A. Marker: None. D.M. Berney: None. W. Tan: None. R. Foo: None. C.A. Mein: None. E. Wozniak: None. A. Sahdev: None. N. Bird: None. K. Laycock: None. E. Adeyeye: None. A. Dawnay: None. D. Gillett: None. A. Prete: None. A.E. Taylor: None. W. Arlt: None. A.N. Bhuva: None. C. Manisty: None. K.J. Cruickshank: None. H. Cheow: None. G. Mark: None. W. Drake: None. M.J. Brown: None. Introduction: A recent prospective, within-patient study (MATCH) demonstrated 11C-metomidate PET-CT is non-inferior to Adrenal Vein Sampling in accurately detecting unilateral Primary Aldosteronism (PA)1. At 6 months post-adrenalectomy 79.5% and 28.2% of patients achieved, respectively, partial/complete or complete clinical success (by PASO consensus). These outcomes reiterate the need for careful selection of patients for surgery, especially when a widely available non-invasive test could increase, by many-fold, the number of patients diagnosed with unilateral PA. In MATCH, age, sex, genotype of tumour and systolic blood pressure (SBP) response (reduction to &amp;lt;135 mmHg) after 4 weeks of Spironolactone therapy all predicted clinical success at 6 month-adrenalectomy. Aims and Methods: To determine whether these short-term clinical outcomes and the above predictors of success are sustained at 2-year follow-up; and establish whether baseline urinary hybrid steroid (18-OH cortisol/cortisol) ratio could assist clinical decision making by providing surrogate evidence of genotype and probability of clinical success. We report outcomes for all patients who had undergone adrenalectomy with ­­&amp;gt;2-year follow-up on 20th December 2022, including all 78/142 surgical patients in the original study, in addition to 18/40 in the study extension. Results: Partial/complete or complete clinical success were achieved in 78/96 (81%) and 24/96 (25%) of patients respectively. The mean defined daily dose of antihypertensives at 2 years was 1.32 (SD 1.76), comparable to 1.34 (1.90) at 6 months, and significantly lower than at baseline: 3.86 (2.47), t = 7.70, p = 0.0001. Younger age and female sex were associated with higher likelihood of complete clinical success (Fisher’s Exact test p = 0.0084 and p = 0.0001 respectively). Pre-operatively, SBP reduction to &amp;lt;135 mmHg after 4 weeks of spironolactone was seen in 10/23 patients and associated with higher likelihood of complete clinical success at 2 years (versus 6/39 complete clinical success in the SBP &amp;gt;135 mmHg group, Fisher’s exact test p = 0.0135). 13/18 (72%) of patients harbouring KCNJ5 mutations achieved complete clinical success at 2 years, compared to 1/20 (5%) with CACNA1D mutations. A baseline hybrid steroid ratio &amp;gt;2 was seen in 13/14 (93%) patients with KCNJ5 mutations. Absence of clinical success in the KCNJ5 cohort was seen in the only patient with a ratio &amp;lt;2. Conclusion: Favorable clinical outcomes seen at 6 months post-adrenalectomy were sustained at 2-year follow-up. SBP response to spironolactone and KCNJ5 genotype are both potential predictors of ongoing clinical success at 2 years. The presence of high urinary hybrid steroid ratios in patients with KCNJ5 mutations could highlight, pre-operatively, those likely to derive the most clinical benefit from surgery. 1. Nature Medicine, in press, https://doi.org/10.1038/s41591-022-02114-5 Presentation: Thursday, June 15, 2023

FRI137 Histologic And Genetic Characteristics Of Primary Aldosteronism In Late Adulthood

Abstract Disclosure: A. Blinder: None. K. Nanba: None. D. Sapiro: None. J. Baker: None. C. Liu: None. I. Bancos: None. M. Lyden: None. W. Young: None. H. Wachtel: None. D. Cohen: None. J. Luther: None. T.A. Williams: None. M. Reincke: None. T. Else: None. T.J. Giordano: None. A. Udager: None. W.E. Rainey: None. Background: There is accumulating evidence that changes in adrenal histology and regulation of renin-angiotensin-aldosterone system occur with age. Primary aldosteronism (PA) is the most common cause of endocrine hypertension. Recent clinical studies suggest unique characteristics of PA in elderly patients, including high prevalence of comorbidities and low clinical cure rate after adrenalectomy. However, histologic and genetic features of PA in late adulthood have not been well elucidated. Objective: To determine histologic and genetic characteristics of PA in late adulthood. Methods: Seventy-six patients who underwent unilateral adrenalectomy at the age of 60 or older were included in this study. Formalin-fixed, paraffin-embedded (FFPE) sections of resected adrenal tissue were used for the analysis. Aldosterone synthase (CYP11B2) immunohistochemistry (IHC) was performed to identify the source of inappropriate aldosterone production. Histologic classification was made according to the HISTALDO (histopathology of primary aldosteronism) consensus. CYP11B2 IHC-guided DNA capture followed by sequencing analysis using direct Sanger sequencing or ion torrent based next-generation sequencing (NGS) was performed. Results: Median age of the studied patients was 70 (interquartile range: 64-73). Based on the CYP11B2 IHC, histologic diagnosis was made as follows; aldosterone-producing adenoma (APA, n=23), aldosterone-producing nodule (APN, n=41), and non-functioning adrenocortical adenoma (NFA, n=6). The remaining six cases had multiple CYP11B2-expressing neoplastic lesions, including APA with APN (n=2) and multiple APNs (n=4). Co-existence of NFA with APA or APN(s) was also observed in 13. Sequencing analysis revealed that somatic KCNJ5 mutation was the most frequent genetic alteration in APA (7/23, 30%), whereas somatic CACNA1D mutation was the most common genetic cause of APN (20/39 with successful sequencing, 51%). Of the 18 sequenced NFA DNA samples, two had somatic mutations in CTNNB1 (n=1) and GNAS (n=1). Conclusion: APN is the major histologic feature of PA in late adulthood. Co-existence of NFA also appears to be a frequent observation in this age population. Somatic CACNA1D and KCNJ5 mutations are common in APN and APA, respectively. Presentation: Friday, June 16, 2023

OR02-05 Differences In Genomic Characteristics Of Small Aldosterone-producing Lesions Between Normal And Pathologic Adrenals

Abstract Disclosure: J. Lim: None. S. Plaska: None. &amp;. Liu: None. J. Baker: None. A. Blinder: None. W.E. Rainey: None. A.M. Udager: None. Introduction: Primary aldosteronism (PA) – the most common form of secondary hypertension – is characterized by inappropriate adrenal aldosterone production under suppressed renin conditions. The majority of PA is caused by unilateral aldosterone-producing adenomas (APA) and bilateral hyperaldosteronism (BHA). Emerging evidence suggests that BHA is associated with the accumulation of small aldosterone-producing lesions (less than 1 cm in diameter), including aldosterone-producing micronodules (APM) and/or aldosterone-producing nodules (APN). However, previous studies have shown that APM may also be observed in normal adrenals, and thus, the impact of APM and APN on the development and progression of aldosterone overproduction is not well established. In this study, we compare the frequency and distribution of somatic aldosterone-driver mutations in small aldosterone-producing lesions from normal and pathologic adrenals. Methods: Twenty-eight normal adrenals from deceased renal donors (n=24) and patients undergoing radical nephrectomy for renal cell carcinoma (n=4) were compared with 24 pathologic adrenals from patients with PA who underwent adrenalectomy at the University of Michigan. CYP11B2 immunohistochemistry-guided capture was performed to selectively obtain APM and APN DNA from formalin-fixed paraffin embedded tissue, and mutation status for all known aldosterone-driver genes was determined by Ion Torrent-based targeted next-generation sequencing. Results: From normal adrenals, 57 APM from 12 female and 16 male subjects (median age = 49 years) were analyzed, and approximately 46% of these lesions harbored an aldosterone-driver mutation. The most commonly mutated gene in normal samples was CACNA1D (n = 23; 40.4%), but no KCNJ5 mutations were identified, and no significant sex differences were observed. From pathologic adrenals, 56 APM and 7 APN from 17 female and 7 male subjects (median age = 46.5 years) were analyzed, and the frequency of aldosterone-driver mutations (approximately 70%) was significantly higher than normal adrenals (p-value &amp;lt; 0.05). Although CACNA1D mutations were still predominant in pathologic adrenals (n = 33; 52.4%), a number of KCNJ5 mutations were also observed (n = 5; 7.9%) – particularly in APN. Finally, there was a higher rate of aldosterone-driver mutations in small aldosterone-producing lesions in pathologic adrenals from men compared to women (93.3% vs. 62.5%; p-value = 0.051). Conclusions: The frequency and distribution of aldosterone-driver mutations in small aldosterone-producing lesions (APM and APN) in pathologic adrenals are different from those in normal adrenals and may also vary by sex. Further research is needed on how these different APM patterns affect autonomous aldosterone production in patients with PA. Presentation: Thursday, June 15, 2023

KCNJ5 mutation is a predictor for recovery of endothelial function after adrenalectomy in patients with aldosterone-producing adenoma.

The relationship of KCNJ5 mutation with vascular function and vascular structure in aldosterone-producing adenoma (APA) patients before and after adrenalectomy remains unclear. The purpose of this study was to evaluate the influence of KCNJ5 mutation on vascular function and vascular structure in APA and the effects of adrenalectomy on vascular function and vascular structure in APA patients with and those without KCNJ5 mutation. Flow-mediated vasodilation (FMD), nitroglycerine-induced vasodilation (NID), brachial artery intima-media thickness (IMT), and brachial-ankle pulse wave velocity (baPWV) were measured to assess vascular function and vascular structure in 46 APA patients with KCNJ5 mutation and 23 APA patients without KCNJ5 mutation and in 69 matched pairs of patients with essential hypertension (EHT). FMD, NID, brachial IMT and baPVW were evacuated before adrenalectomy and at 12 weeks after adrenalectomy in APA patients with KCNJ5 mutation and APA patients without KCNJ5 mutation. FMD and NID were significantly lower in APA patients than in patients with EHT. There was no significant difference in FMD or NID between patients with and those without KCNJ5 mutation. In APA patients with KCNJ5 mutation, FMD and NID after adrenalectomy were significantly higher than those before adrenalectomy. In APA patients without KCNJ5 mutation, only NID after adrenalectomy was significantly higher than that before adrenalectomy. Endothelial function in APA patients with KCNJ5 mutation was improved by adrenalectomy in the early postoperative period. KCNJ5 mutation is a predictor for early resolution of endothelial function by adrenalectomy. This study was approved by principal authorities and ethical issues in Japan (URL for Clinical Trial: http://www.umin.ac.jp/ctr/index.htm Registration Number for Clinical Trial: UMIN000003409).

Identifying KCNJ5 Mutation in Aldosterone-Producing Adenoma Patients With Baseline Characteristics Using Machine Learning Technology

BackgroundPrimary aldosteronism is characterized by inappropriate aldosterone production, and unilateral aldosterone-producing adenoma (uPA) is a common type of PA. KCNJ5 mutation is a protective factor in uPA; however, there is no preoperative approach to detect KCNJ5 mutation in patients with uPA. ObjectivesThis study aimed to provide a personalized surgical recommendation that enables more confidence in advising patients to pursue surgical treatment. MethodsWe enrolled 328 patients with uPA harboring KCNJ5 mutations (n = 158) or not (n = 170) who had undergone adrenalectomy. Eighty-seven features were collected, including demographics, various blood and urine test results, and clinical comorbidities. We designed 2 versions of the prediction model: one for institutes with complete blood tests (full version), and the other for institutes that may not be equipped with comprehensive testing facilities (condensed version). ResultsThe results show that in the full version, the Light Gradient Boosting Machine outperformed other classifiers, achieving area under the curve and accuracy values of 0.905 and 0.864, respectively. The Light Gradient Boosting Machine also showed excellent performance in the condensed version, achieving area under the curve and accuracy values of 0.867 and 0.803, respectively. ConclusionsWe simplified the preoperative diagnosis of KCNJ5 mutations successfully using machine learning. The proposed lightweight tool that requires only baseline characteristics and blood/urine test results can be widely applied and can aid personalized prediction during preoperative counseling for patients with uPA.

18-Oxocortisol: A journey

The search for mineralocorticoids to explain some cases of low renin hypertension with suppressed aldosterone levels led to the isolation of the abundant steroid 18-hydroxycortisol in human urine. 18-Hydroxycortisol proved to be inactive, but because of its similarity to precursors for the synthesis of aldosterone, bullfrog adrenals were incubated with cortisol, resulting in the discovery of 18-oxocortisol which is structurally similar to aldosterone, but with a 17α-hydroxy group like cortisol. 18-Oxocortisol is a weak mineralocorticoid. Its synthesis occurs primarily in the zona glomerulosa where co-expression of the CYP11B2 (aldosterone synthase) and the CYP17A1 (17α-hydroxylase) occurs in a variable number of cells. The clinical value of the measurement of 18-oxocortisol is that it serves to distinguish subtypes of primary aldosteronism. It is significantly elevated in patients with aldosterone-producing adenomas in comparison to those with idiopathic bilateral hyperaldosteronism and helps predict the type of somatic mutation in the aldosterone-producing adenomas, as it is higher in those with KCNJ5 mutations compared to other gene mutations.

Cardiovascular and metabolic characters of KCNJ5 somatic mutations in primary aldosteronism.

Primary aldosteronism (PA) is the leading cause of curable endocrine hypertension, which is associated with a higher risk of cardiovascular and metabolic insults compared to essential hypertension. Aldosterone-producing adenoma (APA) is a major cause of PA, which can be treated with adrenalectomy. Somatic mutations are the main pathogenesis of aldosterone overproduction in APA, of which KCNJ5 somatic mutations are most common, especially in Asian countries. This article aimed to review the literature on the impacts of KCNJ5 somatic mutations on systemic organ damage. PubMed literature research using keywords combination, including "aldosterone-producing adenoma," "somatic mutations," "KCNJ5," "organ damage," "cardiovascular," "diastolic function," "metabolic syndrome," "autonomous cortisol secretion," etc. APA patients with KCNJ5 somatic mutations are generally younger, female, have higher aldosterone levels, lower potassium levels, larger tumor size, and higher hypertension cure rate after adrenalectomy. This review focuses on the cardiovascular and metabolic aspects of KCNJ5 somatic mutations in APA patients, including left ventricular remodeling and diastolic function, abdominal aortic thickness and calcification, arterial stiffness, metabolic syndrome, abdominal adipose tissue, and correlation with autonomous cortisol secretion. Furthermore, we discuss modalities to differentiate the types of mutations before surgery. KCNJ5 somatic mutations in patients with APA had higher left ventricular mass (LVM), more impaired diastolic function, thicker aortic wall, lower incidence of metabolic syndrome, and possibly a lower incidence of concurrent autonomous cortisol secretion, but better improvement in LVM, diastolic function, arterial stiffness, and aortic wall thickness after adrenalectomy compared to patients without KCNJ5 mutations.

S-47-2: DIAGNOSIS OF PRIMARY ALDOSTERONISM USING ARTIFICIAL INTELLIGENCE

Testing for primary aldosteronism (PA) involves an arduous multistep process culminating in either resection of an adrenal in patients identified with unilateral disease after adrenal venous sampling (AVS) or mineralocorticoid receptor blockade in patients without AVS-based evidence of lateralized aldosterone secretion. Multiple difficulties plague each step, including the confirmatory step where immunoassay interferences lead to many false-positive results (1). Mass spectrometry-based steroidomics provides an alternative approach that combined with artificial intelligence might be used for both screening and stratification of patients for therapeutic intervention. The approach may be particularly useful for identification of patients with unilateral disease due to somatic KCNJ5 mutations, who after imaging studies may proceed directly to adrenalectomy without need for confirmatory studies or AVS. To establish this approach, we developed machine learning (ML) models using a retrospective cohort of 462 patients tested for PA, including 139 with unilateral disease of whom 58 had KCNJ5 mutations (2) ML models are now undergoing external validation in a prospective multicenter study (PROSALDO), which has to date enrolled 542 eligible patients. Among these 186 have received follow-up, which has enabled exclusion of disease in 73 and confirmation of unilateral disease in 38 others according to post-surgical biochemical cure. Among the latter, genotyping in 19 patients identified KCNJ5 mutations in nine, all of whom showed post-operative cure and were correctly predicted by ML to have PA with KCNJ5 mutations. Among these nine and four others who were not part of the PROSALDO trial (3) 10 had their adrenals removed without AVS evidence of lateralization. In an interim analysis (with recognition that ML models were likely compromised by incorrect classifications due to immunoassay inaccuracy), among the 542 PROSALDO patients, 138 and 404 were respectively classified with and without PA. According to areas under receiving-operating characteristic curves (AUC), our ML model for screening performed equally poorly (AUC = 0.927) compared to aldosterone:renin ratios (ARR) determined by mass spectrometry (AUC = 0.909) and immunoassay (AUC = 0.895) measurements of aldosterone. Significantly improved performance was achieved by incorporation of plasma potassium and renin with steroidomics-based models (AUC = 0.9511). Results to date indicate promise for ML approaches to both more accurately screen for PA than possible with the ARR and also identify patients with unilateral disease due to KCNJ5 mutations who may directly proceed to surgery without need for confirmation studies or AVS.

PS-BPB06-10: DISTRIBUTION OF CACNA1D SOMATIC MUTATIONS AMONG CYP11B2 IMMUNOHISTOCHEMISTRY-GUIDED SAMPLES OF ALDOSTERONE-PRODUCING ADENOMA (APA)

Objectives: Past studies in Afro-Caribbean primary aldosteronism (PA) patients have found CACNA1D mutations to be the most common aldosterone-driving mutation among their cohort; a striking difference to Caucasian and Oriental cohorts where KCNJ5 mutations are reported to be the most common. However not many studies had used aldosterone synthase (CYP11B2) immunohistochemistry (IHC)-guided sequencing approach of the whole CACNA1D gene, performing instead unguided adenoma sampling and hotspot sequencing. We thus performed CYP11B2 IHC-guided whole gene sequencing on APAs from an Oriental cohort (Malaysia) and a Caucasian cohort (Czech Republic) for comparison. Design and method: Surgically removed adrenals from 100 Malaysian and Czech PA patients with unilateral disease were studied. Genomic DNA was isolated from adenoma FFPE sections that had a positive CYP11B2 IHC staining and somatic mutations analysis were performed by next generation sequencing (NGS) of known aldosterone-driver genes. Predicted pathogenic mutations were verified by Sanger Sequencing. Results: Out of 100 FFPE samples sent for NGS, 36 were excluded due to the quality of the data. In total 58 of 64 APAs (87.5%) had a pathogenic aldosterone-driver somatic mutation. KCNJ5 mutations were the most common (n = 37, 57.8%), followed by CACNA1D (n = 11, 20.3%), ATP1A1 (n = 6, 9.4%), ATP2B3 (n = 3, 4.7%) and CTNNB1 (n = 2, 3.1%) mutations. Of particular interest, one APA sample had two pathogenic somatic mutations, CACNA1D p.Val1373Met and CTNNB1: p.Thr41Ala. SIFT and Polyphen2 scores predicted both mutations to be deleterious. Both mutations have previously been reported separately to cause APAs. The CACNA1D/CTNNB1 mutant APA, was solitary, with homogeneous CYP11B2 expression while negative for CYP17A1 expression. The APA was adrenalectomized from a 53-year-old male Czech patient. The majority of patients with APAs harboring CACNA1D mutations were male (n = 8, 72.7%) and from the Czech cohort (n = 7, 64%). There were no significant differences in age at adrenalectomy between patients harboring a CACNA1D mutant APA (45 ± 3) and those with a non-CACNA1D mutant APA (48 ± 2). Conclusions: Solitary ZG APAs can harbor both a CACNA1D and a CTNNB1 mutation. The prevalence of CACNA1D in this CYP11B2-IHC guided study is consistent with previous results reported in Oriental and Caucasian cohorts. Although CACNA1D is not the top genetic alteration responsible for APAs in these cohorts, CACNA1D may still be a potential key player in idiopathic hyperaldosteronism and aldosterone-producing micronodules. Thus further study is required to characterize the distribution of CACNA1D pathogenic mutations in bilateral PA.

PS-BPB06-9: PREVALENCE OF KCNJ5 MUTANT ALDOSTERONE-PRODUCING ADENOMAS AMONG MALAYSIAN PATIENTS WITH PRIMARY ALDOSTERONISM

Objective: To verify the prevalence of KCNJ5 mutant aldosterone-producing adenomas (APAs) among primary aldosteronism (PA) patients from a tertiary center in Malaysia Design and method: A total of 80 adrenal samples from adrenalectomies performed between 2010 up to 2020 at Hospital Putrajaya, Malaysia were included in this study. Of the adrenals blocks received, DNA samples from 74 APAs identified by CYP11B2 immunohistochemistry (IHC) staining were sent for KCNJ5 mutation analysis. Gender, ethnicity and age at adrenalectomy were compared between genotypes. Data were statistical analyzed using the Fisher Exact test. Results: 28 of the 74 (37.8%) APAs had a KCNJ5 mutation. The most common KCNJ5 variant was G151R (50%), followed by L168R (39.3%) and T158A (10.7%). Of all the mutant APAs, 19/28 (67.9%) were from female PA patients whereas among controls 26/46 (56.5%) were females. No significant association between KCNJ5 mutation status and gender were found. The majority of mutant APAs were from Malay PA patients (n = 17), followed by Chinese PA patients (n = 11) which follows the whole cohort demography. Thus, ethnicity was not found to be significantly associated with KCNJ5 mutation status. In this cohort, no significant differences in age at of adrenalectomy by KCNJ5 mutation status was seen. Adrenalectomy procedure was performed most frequent between the age of 37–46 years old (n = 30), followed by 47–56 (n = 16), 26–35 (n = 12), 57–66 (n = 11) and 67–76 (n = 2); in 3 patients this information was not known. Conclusions: A similar prevalence of KCNJ5 mutant APAs as found in a previous study performed at the National University of Malaysia Medical Centre between 2000 and 2015 verifies the lower prevalence of KCNJ5 mutant APAs in Malaysian PA patients (37.8%) compared to that observed in other Oriental cohorts (up to 70%). Although, the majority of mutant APAs were from female PA patients (67.9%), no gender bias according to KCNJ5 mutation status was found as the majority of PA patients in this cohort were females (60.8%).