Abstract Because blunted expression of the twik-related acid-sensitive K+ channel 2 (TASK-2) is a common feature of aldosterone-producing adenoma (APA) causing primary aldosteronism (PA), we sequenced the promoter region of the TASK-2 gene (KCNK5) in APAs (n = 76), primary hypertensive patients (n = 98), and 20-year-old healthy volunteers (n = 71), searching for variants that could affect expression of this channel. We found TASK-2 promoter mutations in 25% of the APAs: C999T in 6.6%, G595A in 5.3%, G36A in 5.3%, and C562T, Gins468, G265C, C1247T, G1140T, and C1399T in 1.3% each. The C999T mutation was found in only one of the 98 primary hypertensive patients, but mutations were detected also in 12% of volunteers: 4 carried the C999T, 3 G1288C, 1 the G1140T mutation, and 1 the 468ins mutation. After a 16-year follow-up, none of these patients developed hypertension or PA. The effect of C999T mutation was investigated in H295R cells using reporter vectors with the mutated or the wild-type (WT) TASK-2 promoters. TASK-2 gene expression was decreased by 31% ± 18% (P = 0.01) in mutated compared with WT APA. Likewise, in transfected H295R cells, the C999T mutation decreased TASK-2 transcriptional activity by 35% (normalized luciferase signal fold change: 0.65 ± 0.25, P < 0.001). Thus, mutations in the promoter region of the TASK-2 gene can account for the low expression in ∼25% of APAs. As they did not result in hypertension or PA during long-term follow-up in healthy participants, these mutations do not seem to be a factor in causing PA by themselves. The detection of germ-line mutations in the KCNJ5 gene in rare familial forms of primary aldosteronism (PA) provided compelling evidence for a genetic predisposition to PA, the most common cause of endocrine high blood pressure (1). Moreover, multiple seminal discoveries have recently pointed to altered function of ion channels due to gene mutations (KCNJ5, ATP1A1, ATP2B3, CACNA1H, and CACNA1D) (2–4) as a molecular mechanism of PA. By increasing cytosolic Ca2+ levels, dysfunction of these channels, particularly those involved in K+ handling, can account for enhanced constitutive aldosterone secretion in PA (5). However, the rate of the most common and best characterized of such mutations in the KCNJ5 gene is highly heterogeneous in aldosterone-producing adenoma (APA), ranging from 12% in Western countries to 80% in Asia (median 45%), whereas that of the other genes (ATP1A1, ATP2B3, CACNA1H, and CACNA1D) is even lower (from 5% to 15% of the cases) (6). Additional molecular mechanisms in cases that do not have such functional mutations remain, therefore, to be found. The TASK family generates background, or “leak,” K+ currents that are essential for maintaining a negative (≈ −70 mV) resting membrane potential and low cytosolic and mitochondrial Ca2+ levels in zona glomerulosa cells, thus precluding constitutive excess aldosterone production (7). Accordingly, the knockout of TASK channels in mice created phenotypes similar to human sporadic PA and the familial type 1 form (8–12). Moreover, a consistent blunted expression of the two-pore-domain potassium (K2p) channel 2 [twik-related acid-sensitive K+ channel 2 (TASK-2)] at the gene and protein levels was recently reported in APA (13). In about one-third of APAs (30%), the underexpression of TASK-2 could be explained by enhanced expression of microRNA 23 and 34, which were shown to blunt TASK-2 gene expression by binding to the 3′ UTR of the TASK-2 gene (KCNK5) (13). However, in most of the cases, other yet unknown mechanisms can blunt the expression of TASK-2. Hence, we set up this study to investigate if variations in the promoter sequence of the KCNK5 gene can lead to a low expression of TASK-2 in APA. Materials and Methods Participants Adrenocortical tumors from 76 consecutive patients with a diagnosis of APA unequivocally established by the “4 corners criteria” were studied (1). Whenever necessary, treatment with mineralocorticoid receptor antagonists and oral K+ supplementation made the patients normokalemic at the time of adrenalectomy. Ninety-eight essential hypertensive patients and 71 healthy individuals comprising 20-year-old mariners on service on two ships of the Italian National Naval Force recruited in 2001 were also studied. In all patients and healthy volunteers, germ-line DNA was obtained from peripheral blood. All participants provided an informed consent, and the institutional ethics committees approved all procedures. KCNK5 promoter sequencing DNA was extracted from the APA tissues and the buffy coat with a kit (QIAquick DNA purification kit; Qiagen, Milan, Italy) following the manufacturer’s instructions. The promoter region of the KCNK5 gene (coding for TASK-2 channel) was amplified using three sets of primers (Supplemental Table 1) and then sequenced from position –1504 upstream of the ATG site to the +145 site. Polymerase chain reaction was performed on 100 ng DNA using the Expand HiFi PCR System dNTPack kit (Roche, Milan, Italy) in a final volume of 50 µL containing 2.5 mM MgCl2, 2.6 U Taq, and 300 nM primer. After purification with the GenElute PCR Clean Up Kit (Sigma, Milan, Italy), amplicons were submitted to Sanger sequencing at a dedicated facility (BMR, University of Padova). The mutations were also checked in germline DNA in mutated APA, extracting DNA from the peripheral blood buffy coat. All APAs were also checked for mutations in the selectivity filter regions of the KCNJ5 gene as reported (14). TASK-2 gene expression in mutated and wild-type APA The relative expression of the TASK-2 gene in APA vs a control pool of 10 normal adrenocortical tissues was measured using oligomicroarray (15) and compared between the KCNK5-promoter mutated and wild-type (WT) APA. Luciferase reporter assay To test the effect of the C999T, the most frequent mutation found in APA, we designed three different reporter vectors containing the TASK-2 promoter: one with the WT sequence, one carrying the C999T mutation and, as a negative control, one with a mutation in a different position (G/A mapped 200 bp downstream the C999T). These sequences were fused to the coding region of Gaussia luciferase (1.7 kb inserted in SgfI-MluI restriction sites) (GeneCopoeia, Rockville, MD). As a positive control, we used a vector containing the glyceraldehyde 3-phosphate dehydrogenase promoter sequence fused to the Gaussia luciferase gene (GeneCopoeia) and, as normalizer, the secreted alkaline phosphatase. H295R cells were transfected with 2 μg of vectors with the Nucleofector device (Lonza, Milan, Italy) and the Cell Line Nucleofector® Kit R. After nucleofection, cells were kept for 48 hours in culture; the cell medium was then collected to measure the activities of Gaussia luciferase and of secreted alkaline phosphatase. The high-sensitivity protocol of the Dual Luminescence Assay kit (GeneCopoeia) was followed for the luminescent assay; luminescence was read in the Mithras LB940 luminometer (Berthold, Milan, Italy). Nucleofection efficiency was checked by transfecting cells with 2 μg of the pmaxGFP Vector (Cell Line Nucleofector® Kit R; Lonza). Each experiment was done three times in triplicate. The ratio of luminescence intensity of the Gaussia luciferase signal over the secreted alkaline phosphatase signal was calculated to minimize the impact of transfection efficiency variability. Transcription factors binding site prediction The PROMO (16) and the TFBIND (17) software were used for the in silico prediction of transcription factors binding sites in or near to the C999T mutation. Gene expression of transcription factors identified to bind the region of the C999T mutation was checked in the Gene Expression Omnibus profiles database. Statistical analysis For statistical analysis, we used SPSS (version 24 for Mac; SPSS, Inc., Milan, Italy) and the GraphPad Prism (version 6.00 for Mac; GraphPad Software, La Jolla, CA) software. Results KCNK5 promoter sequencing We identified nine different heterozygous mutations in the TASK-2 (KCNK5) promoter region in 25% of APAs (19/76) (Table 1); of them, three recurred in more than one APA: C999T was present in five samples (6.5%) (Fig. 1A), G595A in four (5.2%), and G36A in three samples (3.9%). No APA showed multiple mutations. Germline DNA was sequenced in the 11 available specimens from patients with mutated APA. All variants (Table 1) were germline, except the G1140T, which was only somatic; for the G263C, the germline mutational status could not be verified because the DNA was not available. No other PA cases could be identified in the kinships of mutated patients. Table 1. Summary of the Mutations in the TASK-2 (KCNK5) Promoter Region Detected in APA Mutation Position Nucleotide Change Mutation Rate % Reference SNP Reported Mutation Rate, % a Germline Mutation b High Levels of miR23 or miR34 −36 G→A 3.9 Yes No −263 G→C 1.3 NA No −468 G insertion 1.3 Yes No −562 C→T 1.3 Yes No −595 G→A 5.2 Yes In one case −999 C→T 6.5 rs115955810 0.66 Yes No −1140 G→T 1.3 rs538720853 0.24 No No −1247 C→T 1.3 Yes No −1399 C→T 1.3 Yes No Mutation Position Nucleotide Change Mutation Rate % Reference SNP Reported Mutation Rate, % a Germline Mutation b High Levels of miR23 or miR34 −36 G→A 3.9 Yes No −263 G→C 1.3 NA No −468 G insertion 1.3 Yes No −562 C→T 1.3 Yes No −595 G→A 5.2 Yes In one case −999 C→T 6.5 rs115955810 0.66 Yes No −1140 G→T 1.3 rs538720853 0.24 No No −1247 C→T 1.3 Yes No −1399 C→T 1.3 Yes No Abbreviation: SNP, single-nucleotide polymorphism. a SNP database source. b Mutation detected in at least one of the available germline DNAs. View Large Figure 1. View largeDownload slide (A) Example of sequencing result of an APA with the C999T mutation. (B) TASK-2 gene expression in WT and mutated APA. Gene expression is calculated as percentage of fold change of TASK-2 in APA (n = 28) relative to a pool of 10 normal adrenal cortexes. SEM, standard error of the mean. Figure 1. View largeDownload slide (A) Example of sequencing result of an APA with the C999T mutation. (B) TASK-2 gene expression in WT and mutated APA. Gene expression is calculated as percentage of fold change of TASK-2 in APA (n = 28) relative to a pool of 10 normal adrenal cortexes. SEM, standard error of the mean. A germline C999T mutation was detected in one of the 98 primary hypertensive patients: the patient had had a hemorrhagic stroke 27 years before at age 30 and was on multiple antihypertensive drugs because of stage III hypertension (18). On computed tomography, he had a subcentrimetric lump in the left adrenal gland, but a lateralized aldosterone secretion could not be demonstrated on adrenal vein sampling. Among the 71 healthy volunteers, 12% had a germline mutation in the KCNK5 promoter region: 4 carried the C999T, 3 a new mutation in position G1288C, 1 the G1140T mutation, and 1 the 468ins mutation. None of these subjects, who had been recruited in 2001, developed arterial hypertension or PA after 16 years of follow-up. The C999T (rs115955810) and G1140T (rs538720853) mutations were already reported in the dbSNP (National Center for Biotechnology Information website), but their allele frequency was much higher in our APA cohort (6.5% for C999T and 1.3% for G1140T) than in the public-reported Minor Allele Frequency [0.66% and 0.24%, respectively; 1000 Genomes source (19)]. Molecular and clinical characteristics of mutated and wild-type APA The mutated APA showed a mean TASK-2 gene expression of 31% ± 18% lower than that of the WT APA (P = 0.01) (Fig. 1B). The clinical and biochemical features of patients with or without the KCNK5 promoter mutations showed no significant differences (Table 2). Of the 76 APA, 11 (14.5%) were also mutated in the KCNJ5 gene; of them, 4 carried both TASK-2 and KCNJ5 mutations (3 the G151R and 1 the L168R variants). No obvious differences in the clinical phenotype were detected between patients carrying both TASK-2 and KCNJ5 mutations compared with the WT or single-mutation carriers. Table 2. Comparison Between Patients With APA With and Without TASK-2 Promoter Mutations Characteristic Mutated APA (n = 19) Not Mutated APA (n = 57) Preadrenalectomy Postadrenalectomy Preadrenalectomy Postadrenalectomy Age, mean ± SD, y 48 ± 11 51 ± 13 Sex (% f/m) 62/38 63/37 Systolic blood pressure, mean ± SD, mm Hg 166 ± 12 126 ± 13 156 ± 16 134 ± 16 Diastolic blood pressure, mean ± SD, mm Hg 102 ± 15 81 ± 7 90 ± 14 80 ± 8 Plasma aldosterone concentration, mean ± SD, ng/dL 23.93 ± 7.76 9.94 ± 4.98 20.35 ± 18.23 8.15 ± 4.44 Plasma renin activity, mean ± SD, μg/L/h 0.363 ± 0.200 1.87 ± 2.29 0.752 ± 0.956 1.58 ± 1.94 Characteristic Mutated APA (n = 19) Not Mutated APA (n = 57) Preadrenalectomy Postadrenalectomy Preadrenalectomy Postadrenalectomy Age, mean ± SD, y 48 ± 11 51 ± 13 Sex (% f/m) 62/38 63/37 Systolic blood pressure, mean ± SD, mm Hg 166 ± 12 126 ± 13 156 ± 16 134 ± 16 Diastolic blood pressure, mean ± SD, mm Hg 102 ± 15 81 ± 7 90 ± 14 80 ± 8 Plasma aldosterone concentration, mean ± SD, ng/dL 23.93 ± 7.76 9.94 ± 4.98 20.35 ± 18.23 8.15 ± 4.44 Plasma renin activity, mean ± SD, μg/L/h 0.363 ± 0.200 1.87 ± 2.29 0.752 ± 0.956 1.58 ± 1.94 Abbreviation: SD, standard deviation. View Large In vitro effect of the C999T mutation The luciferase signal in H295 cells transfected for 48 hours with a reporter vector containing the Gaussia luciferase coding gene fused to the TASK-2 mutated (C999T) promoter was 35% lower than in cells transfected with the WT promoter (P < 0.001) (fold change of normalized luciferase signal: 0.65 ± 0.25, of WT promoter sequence P < 0.001) (Fig. 2). The luciferase signal of the negative control carrying a mutation in a different position of the TASK-2 promoter showed no changes compared with the WT promoter sequence (Fig. 2). These findings indicate that only the C999T mutation is functional in that it lowers the transcription of the TASK-2 gene. Figure 2. View largeDownload slide Luciferase reporter assay in H295 cells. Three different reporter vectors were transfected: one containing the WT sequence of the TASK-2 promoter (1.7 kb inserted in SgfI-MluI restriction sites), one carrying the C999T mutation, and one with a mutation in a different position of the TASK-2 promoter. Each sequence was fused to the Gaussia luciferase. To normalize transfection data, the secreted alkaline phosphatase was used as a second reporter gene. Experiments were done three times in triplicate. The ratio of luminescence intensities of the Gaussia luciferase signal over the secreted alkaline phosphatase signal was calculated with the normalized luciferase signal. ns, not significant; SD, standard deviation. Figure 2. View largeDownload slide Luciferase reporter assay in H295 cells. Three different reporter vectors were transfected: one containing the WT sequence of the TASK-2 promoter (1.7 kb inserted in SgfI-MluI restriction sites), one carrying the C999T mutation, and one with a mutation in a different position of the TASK-2 promoter. Each sequence was fused to the Gaussia luciferase. To normalize transfection data, the secreted alkaline phosphatase was used as a second reporter gene. Experiments were done three times in triplicate. The ratio of luminescence intensities of the Gaussia luciferase signal over the secreted alkaline phosphatase signal was calculated with the normalized luciferase signal. ns, not significant; SD, standard deviation. Prediction of transcription factor binding region on the C999T site We performed an in silico prediction of the transcription factors binding site in the WT and mutated sequences to investigate if the functional effect of the C999T mutation was associated with loss of a transcription factor binding site. Both the PROMO (16) and the TFBIND (17) software predicted a consensus sequence for the Stat1 factor from position −997 to −1006 (TTCCCGTAG); moreover, this site was disrupted by the presence of the C999T variant. Of note, the expression of Stat1 was reported in H295 and APA cells in the Gene Expression Omnibus profiles database (GDS3556/209969_s_at/STAT1 and GDS2860/209969_s_at/STAT1, respectively). In position −1291, we identified a response element for nuclear retinoic acid receptors, retinoid X receptors, and liver X receptor (20) (TGAGGTC) that in three healthy volunteers was mutated (G1288C). Discussion The blunted expression of the TASK-2 channel identified as a consistent molecular feature of APA can derive from several potential mechanisms (13, 21). We previously found that the expression of TASK-2 in APA was inversely related with the expression of 13 microRNAs (13), only three of which were predicted to bind the TASK-2 3′ UTR. Moreover, of these microRNAs, two—miR23 and miR34—were shown to significantly blunt the expression of TASK-2 in vitro in a reporter gene assay. An increased expression of miR was, however, found in only 30% of the APAs, which indicated that other mechanisms were implicated. The present results identify one such additional mechanism: by sequencing the TASK-2 gene (KCNK5) promoter region, we identified mutations in 25% of the APAs, of which two (C999T, rs115955810 and G1140T, rs538720853) were already present in the dbSNP database, and seven were novel. The reported allele frequency of the known mutations in the general population in the 1000 Genomes project (19) was consistent with the percentage (1%) of the C999T mutation found in our cohort of primary hypertensive patients but about 10-fold lower than in our APA cohort. To investigate the functional relevance of the most prevalent mutation detected in APA, C999T, we compared the transcriptional activity of the TASK-2 promoter mutant with that of the WT TASK-2 promoter in a reporter in vitro assay. By this approach, we could show that introduction of the C999T mutation significantly decreases the transcription activity of the gene by 35% (Fig. 2), an extent remarkedly similar to the percentage decrease of TASK-2 expression of APA carrying the mutations compared with WT APA. The prediction of the binding site loss for Stat1 transcription factor in the presence of the C999T variant and the reported expression of this factor in both H295 cells and in APA (GDS3556/209969_s_at/STAT1 and GDS2860/209969_s_at/STAT1, respectively) offer a possible mechanism for the decreased in vitro transcriptional activity. It is worth pointing out that most of the KCNK5 promoter variants involved both the APA and the surrounding adrenal cortex because they were germline, thus suggesting that they are a genetic cause of PA and/or hypertension. In contrast with this hypothesis, we did discover these mutations in 12% of healthy normotensive mariners, who remained normotensive and did not develop overt PA after 16 years of follow-up. Thus, these mutations could not be causative of PA per se, and other factors likely need to occur for PA to develop. The latter could comprise somatic mutations in other genes involved in the hyperaldosteronism in APA as, for example, KCNJ5. Interestingly, four of our patients with mutated APA have also a somatic functional variant of the KCNJ5 gene (three the G151R and one the L168R), which, however, seemingly did not markedly change their clinical phenotype. In this cohort of APA, the frequency of KCNJ5 mutations was lower than that found in a recent large meta-analysis (median 43%; range, 12% to 80%) (6), possibly reflecting the occurrence of other, yet unknown, gene variants or a selection bias. This could be because with the systematic screening of hypertensive patients for PA exploited in our center, we pinpoint the less florid cases, whereas KCNJ5 mutations are held to imply a more florid clinical phenotype (6). Thus, the low expression of TASK-2 can be associated with different genetic and epigenetic predisposition factors, as sketched in Fig. 3. Of note, an additional novel mutation (G1288C) in the binding site for the retinoid X and retinoic acid receptors, which in a cell model were recently shown to regulate aldosterone production (22), was not found in any of our APAs but was detected in three healthy mariners, who remained normotensive for 16 years. Thus, whether this mutation could play a functional role, either promoting PA or protecting from it, remains to be explored. Figure 3. View largeDownload slide Schema of proposed mechanisms causing TASK-2 low levels in APA. Different mechanisms may result in the TASK-2 low expression that features all APAs. This may be due to genetic causes (mutations in TASK-2 promoter), epigenetic factors (high levels of microRNA hsa-miR23 and hsa-miR34), or as-yet-unknown conditions. Figure 3. View largeDownload slide Schema of proposed mechanisms causing TASK-2 low levels in APA. Different mechanisms may result in the TASK-2 low expression that features all APAs. This may be due to genetic causes (mutations in TASK-2 promoter), epigenetic factors (high levels of microRNA hsa-miR23 and hsa-miR34), or as-yet-unknown conditions. As for mutations in other genes associated with PA, the TASK-2 gene (KCNK5) variants were detected in only 25% of the APAs, in agreement with the current view that they are highly heterogeneous tumors (15, 23). Although the mechanisms causing TASK-2 low expression in nonmutated samples remain to be clarified, the finding that 30% of APAs have high levels of two microRNAs (hsa-miR-23 and hsa-miR-34) and that these microRNAs are able to cause the degradation of the TASK-2 transcript (13), together with the present results, collectively suggest that different molecular mechanisms can eventually result in the same molecular feature (e.g., low expression of TASK-2 with ensuing aldosteronism) (Fig. 3). Of note in our present series, only one subject had high levels of hsa-miR-23 and hsa-miR-34 and a mutation in the TASK-2 promoter (Table 1). Conclusions In summary, this study shows that in about one-fourth of APAs, the low expression of TASK-2 is associated with functional mutations in the promoter region of the TASK-2 gene (KCNK5). These variants could thus act as predisposing factors that, when associated with other genetic variations acquired by the adrenal gland (e.g., somatic mutations in other genes involved in the autonomous production of aldosterone) and/or with environmental factors, can eventually coincide with the development of PA in a relevant proportion of the cases. Abbreviations: APA aldosterone-producing adenoma PA primary aldosteronism TASK-2 twik-related acid-sensitive K+ channel 2 WT wild-type. Acknowledgments Financial Support: This study was supported by research grants from the Young Research Program of Italy’s Health Ministry (Project GR-2009-1524351 to L.L.), the International PhD Program in Arterial Hypertension and Vascular Biology of the University of Padova (to G.P.R.), and FORICA (The Foundation for Advanced Research in Hypertension and Cardiovascular Diseases) (to G.P.R.). Disclosure Summary: The authors have nothing to disclose. References 1. 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; PAPY Study Investigators. A prospective study of the prevalence of primary aldosteronism in 1,125 hypertensive patients. J Am Coll Cardiol . 2006; 48( 11): 2293– 2300. Google Scholar CrossRef Search ADS PubMed 2. 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Published: Mar 1, 2018
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