Somatic ATP1A1, ATP2B3, and KCNJ5 Mutations in Aldosterone-Producing AdenomasNovelty and Significance
Aldosterone-producing adenomas (APAs) cause a sporadic form of primary aldosteronism and somatic mutations in the KCNJ5 gene, which encodes the G-protein–activated inward rectifier K+ channel 4, GIRK4, account for ≈40% of APAs. Additional somatic APA mutations were identified recently in 2 other genes, ATP1A1 and ATP2B3, encoding Na+/K+-ATPase 1 and Ca2+-ATPase 3, respectively, at a combined prevalence of 6.8%. We have screened 112 APAs for mutations in known hotspots for genetic alterations associated with primary aldosteronism. Somatic mutations in ATP1A1, ATP2B3, and KCNJ5 were present in 6.3%, 0.9%, and 39.3% of APAs, respectively, and included 2 novel mutations (Na+/K+-ATPase p.Gly99Arg and GIRK4 p.Trp126Arg). CYP11B2 gene expression was higher in APAs harboring ATP1A1 and ATP2B3 mutations compared with those without these or KCNJ5 mutations. Overexpression of Na+/K+-ATPase p.Gly99Arg and GIRK4 p.Trp126Arg in HAC15 adrenal cells resulted in upregulation of CYP11B2 gene expression and its transcriptional regulator NR4A2. Structural modeling of the Na+/K+-ATPase showed that the Gly99Arg substitution most likely interferes with the gateway to the ion binding pocket. In vitro functional assays demonstrated that Gly99Arg displays severely impaired ATPase activity, a reduced apparent affinity for Na+ activation of phosphorylation and K+ inhibition of phosphorylation that indicate decreased Na+ and K+ binding, respectively. Moreover, whole cell patch-clamp studies established that overexpression of Na+/K+-ATPase Gly99Arg causes membrane voltage depolarization. In conclusion, somatic mutations are common in APAs that result in an increase in CYP11B2 gene expression and may account for the dysregulated aldosterone production in a subset of patients with sporadic primary aldosteronism.
- adrenal glands
- Conn adenoma
- potassium channels
- sodium-potassium–exchanging ATPase
See Editorial Commentary, pp 24–26
Primary aldosteronism (PA) is the most common form of secondary hypertension, with a prevalence of 5% to 15% among hypertensive patients and is characterized by the autonomous hypersecretion of aldosterone. Sporadic PA and 3 familial forms (familial hyperaldosteronism types I, II, and III) have been described.1 Sporadic PA accounts for >90% of all cases and is caused by either an aldosterone-producing adenoma (APA), which can be surgically removed, or bilateral adrenal hyperplasia, which is treatable with mineralocorticoid receptor antagonists.
Somatic APA mutations in the KCNJ5 gene, which encodes the G-protein–activated inward rectifier K+ channel 4, GIRK4 (also called the inward rectifier K+ channel, Kir3.4), were first identified by Choi et al.2 Subsequently, Boulkroun et al3 determined a 34% prevalence of KCNJ5 mutations in a large European cohort of 380 APA. Intriguingly, the KCNJ5 mutations were markedly more prevalent in women,3 and this predominance was confirmed by successive studies.4,5 To date, 5 different KCNJ5 mutations causing sporadic PA have been identified, the majority of which are the substitutions p.Gly151Arg or p.Leu168Arg2,3 or, more rarely, p.Glu145Gln,5 p.Thr158Ala,6 and p.Ile157del.7 A common feature of all the mutations is that they are situated in or around the selectivity filter of the K+ channel pore and in several cases have been shown to cause a loss of channel selectivity resulting in sodium entry and membrane depolarization and the opening of voltage-gated Ca2+ channels.2,7,8 The resultant increase in the intracellular Ca2+ concentration determines an increase in the transcription of the CYP11B2 gene, which encodes aldosterone synthase, and in the production of aldosterone.4,8 Furthermore, APAs harboring KCNJ5 mutations have been reported to display higher levels of CYP11B2 expression compared with those with wild-type KCNJ5,4,9 although this was not confirmed in another study.10
To identify further genetic determinants of PA, Beuschlein et al11 performed exome sequencing of APAs from male patients without somatic KCNJ5 mutations. Novel mutations in ATP1A1, encoding the Na+/K+-ATPase α-subunit, and in ATP2B3, encoding the plasma membrane Ca2+-ATPase 3, were identified. In a European cohort of 308 APAs, the prevalence of these mutations in ATP1A1 and ATP2B3 was 5.2% and 1.6%, respectively. The ATP1A1 mutations were primarily c.311T>G (p.Leu104Arg), less frequent was a c.995T>G mutation (p.Val332Gly), as well as 2 different in-frame DNA deletions that result in the same deletion at the amino acid level (p.Phe100_Leu104del). In ATP2B3, 3 different in-frame DNA deletions were identified that result in either p.Leu425_Val426del or p.Val426_Val427del. In all cases, the affected amino acids are highly conserved across species and between different members of the P-type ATPase family. In vitro functional characterization of the substitutions in Na+/K+-ATPase 1 demonstrated that they have a profound effect on K+ binding and ATPase activity and cause membrane depolarization in whole cell patch-clamp studies that in adrenal cells would lead to the opening of voltage-dependent Ca2+-channels as for the KCNJ5 mutations.11
In this study, we have analyzed 112 APAs, diagnosed and surgically removed in Italian referral hypertension centers, for mutations in ATP1A1, ATP2B3, and KCNJ5 and searched for new mutations within known hotspots for genetic alterations in these genes.
An expanded Methods section is available in the online-only Data Supplement.
The protocol was approved by our local ethics committee, and all participants gave their written informed consent.
RNA Isolation and Reverse Transcription Polymerase Chain Reaction
Adrenal tissues were homogenized in 1 mL TRI reagent for RNA extraction, and first-strand cDNA was synthesized from total RNA (5 μg) as described.14
DNA Sequencing of KCNJ5, ATP1A1, and ATP2B3
Polymerase chain reaction (PCR) primers used to amplify cDNA fragments for direct sequencing of ATP1A1 and ATP2B3 are shown in Table S1 in the online-only Data Supplement. The validity of novel mutations was confirmed by sequencing both strands of an independently amplified PCR fragment. The present cohort constitutes 112 APAs from different centers and included 32 samples from the Beuschlein study.11 A full description of these samples is included in the online-only Data Supplement. No mutations were identified in paired peripheral blood DNA samples (n=43), and mutations were absent from paired peritumoral tissue (n=23 of which 7 carried KCNJ5 mutations in the corresponding APA).
KCNJ5 and ATP1A1 Expression Vectors
The rat Atp1a1 cDNA was used for this study so that endogenous human ATP1A1 in the HAC15 cell line could be inactivated by RNA interference using the strategy described.11 For clarity, throughout this article, when referring to either rat or human ATP1A1, the numbering used to describe mutations follows the human cDNA or amino acid numbering.
Modeling of Protein Structures
Structural models of Na+/K+-ATPase 1 (PDB code 2ZXE) were prepared using PyMOL software (www.pymol.org).
Cell Transfection and Gene Silencing
HAC15 cells were grown in culture4 and transfected with plasmids and siRNAs by Amaxa nucleofection using program X005.9 Equivalent transfection rates of rat cDNAs and silencing of HAC15 ATP1A1 were confirmed by real-time TaqMan PCR.
TaqMan Gene Expression Assays
Gene expression levels in transfected cells and in adrenal tissues were determined by real-time PCR using TaqMan gene expression assays using GAPDH as the endogenous reference gene.14 Gene expression levels in APA were compared with those in corresponding surrounding tissue (peri-APA) for 7 of 8 APA with ATP1A1 or ATP2B3 mutations and for 11 APA without mutations in KCNJ5, ATP1A1, or ATP2B3.
Na+/K+-ATPase Functional Assays
COS cells were transiently transfected with rat wild-type or mutated Atp1a1 encoding Na+/K+-ATPase p.Gly99Arg together with a siRNA to interfere specifically with the expression of the endogenous COS cell Atp1a1.11 ATPase activity assays, Na+ dependence of phosphorylation by MgATP and K+ inhibition of phosphorylation by MgATP, were as described.11
Electrophysiological Characterization of Cells Expressing Na+/K+-ATPase Gly99Arg
The full-length cDNA encoding wild-type rat Atp1a1 and the mutated Atp1a1 c.295G>A (p.Gly99Arg) were subcloned into the bicistronic pIRES-CD8 expression vector.15 Human embryonic kidney cells were transfected transiently with Lipofectamine, anti-CD8–labeled Dynabeads were used to identify transfected cells, and patch-clamp whole cell recordings were performed using an EPC-10 amplifier without leak subtraction.11
All data are expressed as mean±SEM for normally distributed variables and as median (25th–75th percentile) for non-normally distributed variables. The Student t test or the Kruskal–Wallis test was used for quantitative variables, and the χ2 test or the Fisher exact test was applied for qualitative variables. Confidence intervals were calculated for frequency data using Wilson’s method.16
Prevalence of Somatic APA Mutations in ATPases
Sequencing analysis of 112 APA DNA samples collected from 5 different Italian centers demonstrated a prevalence of ATP1A1 mutations of 6.3% (Table 1). A novel ATP1A1 c.295G>A mutation (p.Gly99Arg) was identified in a single sample, and the ATP1A1 c.311T>G mutation (p.Leu104Arg)11 was present in 6 samples (Figure 1A and B; Table 1). The c.995T>G mutation (p.Val332Gly) and the deletion mutations (p.Phe100_Leu104del) described previously11 were not detected. However, 1 APA with an ATP2B3 mutation (c.1272-1277delGCTGGT corresponding to p.Leu425-Val426del) was identified (Figure 1C; Table 1). Together, the mutations in these 2 ATPases were present in 7.2% of APAs and were absent from the corresponding peripheral blood DNA, thereby demonstrating their somatic nature. This prevalence is in close agreement with the 6.9% frequency reported by Beuschlein et al,11 but there was no correlation with increased preoperative aldosterone concentrations or with reduced serum K+ levels (Table S2).
Novel Somatic APA Mutation in ATP1A1
The novel APA mutation in ATP1A1 was identified in the Torino 1 group of samples, ATP1A1 c.295G>A (p.Gly99Arg). The patient harboring the p.Gly99Arg mutation displayed a particularly severe form of PA with very low serum potassium (1.4 mEq/L), and the phenotype of this patient is described in the online-only Data Supplement.
Novel Somatic APA Mutation in KCNJ5
Sequence analysis of KCNJ5 demonstrated the presence of 4 different somatic APA mutations in this sample set: the frequently reported c.451G>A/C (p.Gly151Arg) and c.503T>G (p.Leu168Arg) substitutions,2,3 c.472A>G (p.Thr158Ala)6 and a novel c.376T>C (p.Trp126Arg) mutation that affects a highly conserved residue (Figure 1D; Table 2; and Figure S1). In agreement with other reports,3,5 the p.Gly151Arg substitution was identified more frequently than p.Leu168Arg (31.5% versus 9.0%; P<0.02). A wide variation in prevalence was observed between different centers (20.0%–47.6%), but the overall prevalence was 39.3%. There was no correlation with adenoma size, age at diagnosis, or preoperative aldosterone and K+ levels (Table S2). The phenotype of the patient harboring the p.Trp126Arg mutation is described in the online-only Data Supplement.
Prevalence of APA Mutations According to Sex
In this study, the ATPase mutations were distributed equally between men and women (P=0.5), rather than predominantly in men.11 In accordance with previous reports,3–5 the KCNJ5 mutations were more prevalent in women than in men (50.0% APA women carried KCNJ5 mutations versus 29.3% men; P=0.02; Table 3).
Structural Effects of the Na+/K+-ATPase Gly99Arg Mutation
Gly99 resides in the transmembrane domain M1 (Figure 1E and 1F) in close proximity to residues Ile292 and Glu334 in domains M3 and M4, respectively (Figure 1F). The Gly99-Ile292 pair functions as a pivot for the movement of M1 leading to the opening of the gate at the entrance to the cation binding pocket, whereas Glu334 is part of the gate17 (Figure 1F). Introduction of the large positively charged arginine side-chain is likely to cause a severe structural alteration affecting surrounding residues including Glu334 that binds K+ (Figure 1G).
APAs Carrying ATP1A1 and ATP2B3 Mutations Display Increased CYP11B2 Gene Expression
Analysis of gene expression levels by TaqMan real-time PCR demonstrated that the expression of CYP11B2 in APAs with ATPase mutations (in either ATP1A1 or ATP2B3) compared with corresponding peri-APA tissue was significantly higher than that in APAs without mutations in either KCNJ5, ATP1A1, or ATP2B3 (51.6- versus 4.03-fold; P<0.027). In contrast, there was no significant difference in CYP11B1 gene expression (Figure 2).
p.Gly99Arg and p.Leu104Arg Mutations in Na+/K+-ATPase 1 Increase CYP11B2 Expression In Vitro
Expression of rat Atp1a1.c.311T>G and c.295G>A (p.Leu104Arg and p.Gly99Arg, respectively) in HAC15 adrenal cells, in which endogenous ATP1A1 had been silenced, resulted in 1.58±0.10-fold and 1.89±0.07-fold increases in CYP11B2 gene expression, respectively, compared with control cells transfected with empty vector and a control siRNA (P<0.01; Figure 3A). Expression of the p.Gly99Arg mutation also resulted in a 1.28±0.06-fold increase (P<0.01) in the expression of NR4A2, a transcriptional regulator of CYP11B218; in contrast, p.Leu104Arg expression had no statistically significant effect on NR4A2 (Figure 3A).
GIRK4 p.Trp126Arg Mutation Increases CYP11B2 and NR42A Expression In Vitro
Expression of GIRK4 p.Trp126Arg (KCNJ5 c.376T>C) in HAC15 cells resulted in a 1.90±0.18-fold increase in the expression of CYP11B2 compared with control cells and a 4.70±0.73-fold increase in the expression of NR4A2 (P<0.01; Figure 3B). Expression of the most frequent GIRK4 mutation in this study, Gly151Arg, resulted in 5.19±0.61- and 16.78±2.39-fold increases (P<0.01) in CYP11B2 and NR4A2 gene expression, respectively (Figure 3B).
Na+/K+-ATPase Gly99Arg Mutation Displays Altered Functional Activity
The maximal Na+/K+-ATPase activity of Gly99Arg was compared with both the wild-type and a nonphosphorylatable inactive mutant (Asp376Asn). The ATPase activity of Gly99Arg was impaired severely and displayed levels of activity comparable with those of the inactive mutant, which represent background detection (Figure 4A). In addition, the Gly99Arg mutant displayed a reduced affinity for Na+ activation of phosphorylation by MgATP with a half maximal activation at 1.19 mmol/L Na+ compared with 0.58 mmol/L for the wild-type Na+/K+-ATPase (Figure 4B). In the Na+/K+ exchange mechanism, the ATPase is dephosphorylated after K+ binding. Therefore, ATPase phosphorylation is inhibited by K+, which in vitro can be used for measuring K+ affinity. Accordingly, the K+ inhibition of Gly99Arg phosphorylation by MgATP was compared with that of wild-type Na+/K+-ATPase. Gly99Arg was markedly less sensitive to inhibition of phosphorylation by K+ with half maximal inhibition occurring at 0.62 mmol/L K+ compared with 0.074 mmol/L K+ of the wild-type ATPase (Figure 4C).
Na+/K+-ATPase Gly99Arg Mutation Causes Membrane Voltage Depolarization
The effect of the Na+/K+-ATPase Gly99Arg mutation on the membrane voltage of transfected human embryonic kidney cells was measured by whole cell patch-clamp. The membrane voltage was depolarized in cells expressing the Gly99Arg mutation compared with those expressing the wild-type ATPase (Figure 5). The difference in membrane voltage was also observed when Na+ was removed from the bath indicating that the depolarization was not caused by an increased Na+ conductance in transfected cells, but rather by a disturbed ionic gradient and/or loss of net charge transport in cells expressing p.Gly99Arg.
Mutations in the KCNJ5 gene, which encodes the GIRK4 K+ channel, have been identified that cause both sporadic and familial forms of PA.1–3 The mutations are located around the selectivity filter of the K+ channel and cause a loss of ion selectivity and cell depolarization that in adrenal glomerulosa cells result in the opening of voltage-gated Ca2+ channels.2,6,8 The Ca2+ influx stimulates both CYP11B2 and NR4A2 (a transcriptional regulator of CYP11B2) gene expression, and subsequently aldosterone secretion is increased.4,8 In this study, we report a novel somatic mutation in KCNJ5 (GIRK4 p.Trp126Arg), and, therefore, a total of 6 different somatic KCNJ5 mutations have been identified to date with the most prevalent being p.Gly151Arg and p.Leu168Arg. The p.Trp126Arg mutation affects a tryptophan residue that is conserved across species and in close proximity to residues Pro128 and Cys129 that form hydrogen bonds with Thr158,2 a residue mutated to an Ala in both familial hyperaldosteronism type III2 and in sporadic APA.6 We show that overexpression of GIRK4 p.Trp126Arg in adrenal cells results in an increase in CYP11B2 and NR4A2 gene expression as described for other GIRK4 mutations.4,8
Beuschlein et al11 recently reported several newly described somatic APA mutations in 2 different genes, ATP1A1 and ATP2B3, that encode Na+/K+-ATPase 1 and Ca2+-ATPase 3, members of the P-type family of ATPases. In their sample set of 308 APAs, a combined prevalence of 6.8% ATP1A1 and ATP2B3 mutations and 38.3% KCNJ5 mutations was reported. The data from the present study are in close agreement with these observations: in our 112 tumor samples, which included 32 samples from the Beuschlein study,11 we describe a prevalence of 7.2% ATP1A1 and ATP2B3 mutations and 39.3% KCNJ5 mutations. In both studies, concomitant mutations within the same tumor were not observed. However, we found that the ATPase mutations were distributed equally between men and women, although our sample size of 8 APAs with ATPase mutations is smaller than that of the previous study,11 which included 21 ATPase mutations.
Furthermore, we did not find a correlation of KCNJ5 or ATP1A1/ATP2B3 mutational status with clinical characteristics related to adenoma size or disease severity (higher preoperative aldosterone levels and lower serum potassium levels) that has been observed in some studies11,19 but not in others.2,3,5,20 Differences in clinical and biochemical characteristics of patients with or without somatic APA mutations, as well as the prevalence of mutations in different cohorts, could be determined by ethnicity and by patient selection and diagnostic strategies.
For each ATP hydrolyzed, the Na+/K+-ATPase couples the extrusion and uptake of 3 cytoplasmic Na+ for 2 extracellular K+ ions across the plasma membranes of most higher eukaryotes.21,22 This ion transport pathway comprises a channel on each side of the lipid bilayer that access the ion binding pocket.22 The Na+/K+-ATPase Leu104 residue is located in transmembrane domain M1 and positions Glu334 that functions in the binding and occlusion of potassium ions.23 The p.Leu104Arg substitution identified in APAs causes a severe disturbance of K+ binding and ATPase activity resulting in depolarization of the cell membrane.11 In glomerulosa cells, the inactivation of the Na+/K+-ATPase α-subunit by the inhibitor ouabain has been reported to cause a nifedipine-sensitive elevation in the cytoplasmic Ca2+ concentration and a stimulation of aldosterone production.24,25 The novel Na+/K+-ATPase p.Gly99Arg mutation identified in this study is located in transmembrane α-helix M1, intriguingly in the same region as the p.Leu104Arg mutation, in close proximity to residues Ile292 and Glu334, which function in gating entry to the cation binding pocket. The p.Gly99Arg mutation is likely to cause a steric clash with an impact on surrounding residues, including Glu334 that binds K+. Consistently, we show that the p.Gly99Arg mutant displayed minimal ATPase activity, reduced Na+ affinity, and markedly reduced K+ affinity when compared with the wild-type ATPase as observed in phosphorylation studies. These results are in agreement with a disturbed gating mechanism, in particular, affecting K+ binding to the mutated pump. Adrenal zona glomerulosa cells display a distinctive high resting K+ conductance that maintains the high negative membrane potential (−80 mV) of these cells.26 We demonstrate by whole cell patch-clamp studies that the p.Gly99Arg mutation results in the depolarization of the membrane voltage. In adrenal glomerulosa cells, this depolarization results in the opening of voltage-gated Ca2+ channels.2,8 In accordance with an increase in Ca2+ influx, we show that the overexpression of p.Gly99Arg or p.Leu104Arg in adrenal cells results in an increase in CYP11B2 gene expression and also, for the p.Gly99Arg mutation, NR4A2. Finally, the expression of CYP11B2 in APAs harboring ATPase mutations was significantly higher compared with those without these or GIRK4 mutations; an effect of heterozygous ATPase mutations on glomerulosa cells is consistent with the unique sensitivity of these cells to small changes in intracellular K+ and membrane potential.26,27 Taken together, these observations are in agreement with a pathophysiological link between the presence of these mutations and the dysregulated aldosterone secretion in patients with APA.
Genetic alterations that lead to cell depolarization and increased transcription of the CYP11B2 gene in adrenal cells account for a large proportion of APAs (47%). The majority of these mutations affect the GIRK4 K+ channel, whereas a far smaller number alter Na+/K+-ATPase 1 and Ca2+-ATPase 3. In general, the mutations cluster around hotspot regions and in this study we report a rare Na+/K+-ATPase p.Gly99Arg mutation that affects the same protein domain as the more frequent p.Leu104Arg mutation involved in gating of the ion transport pathway. The understanding of new molecular pathways responsible for the dysregulation of aldosterone production may lead to the development of novel therapeutic targets aimed at treating the most common form of secondary hypertension.
Sources of Funding
This study was funded by grants from MIUR 2012 (ex 60%; Ministry of Instruction, University, and Research, Italy; to P. Mulatero); the Danish Medical Research Council, the Novo Nordisk Foundation (Vilhelm Pedersen og Hustrus Legat), and the Lundbeck Foundation (to B. Vilsen); the Deutsche Forschungsgemeinschaft (FOR1086 to R. Warth); “Fondo per gli Investimenti della Ricerca di Base Accordi di Programma 2011” RBAP1153LS-02 from the Ministry of Education, University, and Research, Rome, Italy (to F. Fallo); the Deutsche Forschungsgemeinschaft (Re 752/17-1, to F. Beuschlein and M. Reincke); the Else Kröner-Fresenius-Stiftung for the German Conn’s Registry-Else-Kröner Hyperaldosteronism Registry (to M. Reincke).
The online-only Data Supplement is available with this article at http://hyper.ahajournals.org/lookup/suppl/doi:10.1161/HYPERTENSIONAHA.113.01733/-/DC1.
- Received May 17, 2013.
- Revision received June 3, 2013.
- Accepted September 11, 2013.
- © 2013 American Heart Association, Inc.
- Choi M,
- Scholl UI,
- Yue P,
- et al
- Boulkroun S,
- Beuschlein F,
- Rossi GP,
- et al
- Mulatero P,
- Tauber P,
- Zennaro MC,
- et al
- Williams TA,
- Monticone S,
- Crudo V,
- Warth R,
- Veglio F,
- Mulatero P
- Mulatero P,
- Bertello C,
- Rossato D,
- Mengozzi G,
- Milan A,
- Garrone C,
- Giraudo G,
- Passarino G,
- Garabello D,
- Verhovez A,
- Rabbia F,
- Veglio F
- Funder JW,
- Carey RM,
- Fardella C,
- Gomez-Sanchez CE,
- Mantero F,
- Stowasser M,
- Young WF Jr.,
- Montori VM
- Williams TA,
- Monticone S,
- Morello F,
- Liew CC,
- Mengozzi G,
- Pilon C,
- Asioli S,
- Sapino A,
- Veglio F,
- Mulatero P
- Fink M,
- Lesage F,
- Duprat F,
- Heurteaux C,
- Reyes R,
- Fosset M,
- Lazdunski M
- Einholm AP,
- Toustrup-Jensen M,
- Andersen JP,
- Vilsen B
- Nogueira EF,
- Xing Y,
- Morris CA,
- Rainey WE
- Azizan EA,
- Lam BY,
- Newhouse SJ,
- Zhou J,
- Kuc RE,
- Clarke J,
- Happerfield L,
- Marker A,
- Hoffman GJ,
- Brown MJ
- 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
- Einholm AP,
- Andersen JP,
- Vilsen B
Novelty and Significance
What Is New?
Somatic mutations in the KCNJ5, ATP1A1, and ATP2B3 genes are present in aldosterone-producing adenomas (APAs).
We report a high prevalence (47%) of somatic APA mutations in Italian referral centers for hypertension.
Two novel somatic APA mutations, in KCNJ5 (p.Trp126Arg) and in ATP1A1 (p.Gly99Arg), were identified.
What Is Relevant?
APA-harboring ATP1A1 or ATP2B3 mutations express higher levels of CYP11B2 compared with those without these or KCNJ5 mutations.
A novel ATP1A1 mutation (p.Gly99Arg) displays markedly reduced K+ binding and causes cell depolarization.
Overexpression of ATP1A1 mutations (p.Gly99Arg and p.Leu104Arg) in adrenal cells result in CYP11B2 upregulation.
Somatic APA mutations result in cell depolarization and an increase in CYP11B2 gene expression.