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Departments of Diabetes and Endocrinology (H.T., T.M., T.S., J.T.), Urology (Y.T., T.D.), and General Medicine (H.M.), Gifu University School of Medicine, Yanagido, Gifu 501-1194, Japan; Gifu Red Cross Hospital (M.I.), Iwakura-cho, Gifu 502-8511, Japan; Matsunami General Hospital (N.Y., K.Y.), Kasamatsu, Gifu-Prefecture 501-6062, Japan; and Division of Pediatric Endocrinology, University of Texas Southwestern Medical Center (P.C.W.), Dallas, Texas 75390-9063
Address all correspondence and requests for reprints to: Dr. Tomoatsu Mune, Department of Diabetes and Endocrinology, Room 2S15, Gifu University, 1-1 Yanagido, Gifu 501-1194, Japan. E-mail: mune{at}cc.gifu-u.ac.jp.
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Abstract |
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Objective: The objective of this study was to correlate CYP11B2 genotype with gene expression in adrenal tissue.
Design: We measured mRNA levels of CYP11B2 [presented as a ratio against glyceraldehyde-3-phosphate dehydrogenase (B2/G)] and CYP11B1 in relation to the K173R polymorphism.
Subjects: We studied 28 subjects with aldosterone-producing adenomas (APA) and 18 subjects with normal adrenals.
Main Outcome Measure: The main outcome measure was CYP11B2 expression levels.
Results: Preoperative treatment with spironolactone or ß-blocker in five APA patients was associated with higher B2/G. The B2/G and B2/B1 ratios were much higher even in the remaining 23 APA patients than in subjects with normal adrenals. The B2/G and B2/B1 ratios in normal adrenals and APA were higher in the KK genotype than in the RR genotype. In patients with APA, urinary aldosterone excretion was higher in those with the KK genotype than in those with the KR genotype. Measurement of cDNA band intensities from normal and APA samples of the KR genotype revealed that the R173 allele was transcribed at levels 46.6 ± 12.2% (mean ± SD; n = 7) and 49.1 ± 20.8% (n = 6), respectively, those of the K173 allele.
Conclusions: A CYP11B2 haplotype including –344T and K173 is associated with higher gene expression than the –344C/R173 haplotype, supporting reported associations of –344T with higher aldosterone production and blood pressure.
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Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Subjects and Methods |
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We examined 28 patients with APAs admitted to our department or related hospitals who underwent unilateral adrenalectomy during 1992–2004. They had hypertension, hypokalemic alkalosis, elevated plasma aldosterone concentrations, and suppressed plasma renin activity (Table 1
). Basal blood was collected in 20 patients without medication, six patients receiving calcium antagonists (no. 15, 17, 20, 21, 25, and 26), and two patients receiving calcium antagonist plus 
-adrenergic blockers (no. 12 and 27). After demonstration of laterality of aldosterone secretion by adrenal vein sampling, four patients (no. 1, 2, 3, and 13) were treated with spironolactone until a few days before surgery. One patient (no. 4) with concurrent liver damage received a ß-adrenergic blocker due to suspicious angina pectoris. Signs and symptoms of mineralocorticoid excess improved after tumor resection in all patients. Adrenal adenomas causing overt Cushing’s syndrome (n = 17) were included as another representative group of adrenocortical tumors. As controls, normal adult adrenal tissues (n = 18) were obtained during surgical removal for renal cell carcinomas and cut radially so that cortex and medulla were included in similar proportions. All adrenal tissues were obtained at surgery after patients gave written informed consent. Samples were confirmed as having the expected pathological findings and were stored at –80 C immediately after tumor resection until total RNA was extracted. Tumor volumes were calculated from measured three-dimensional diameters by estimating each tumor as an ellipsoid. The study protocol was approved by the ethical committee of Gifu University School of Medicine.
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Total RNA was prepared from frozen tissues using Isogen (Nippon Gene, Toyama, Japan), and 2.2 µg total RNA was treated with deoxyribonuclease I (Invitrogen Life Technologies, Inc., Gaithersburg, MD) and reverse transcribed with 160 U SuperScript II reverse transcriptase (Invitrogen Life Technologies, Inc.) in a 20-µl reaction volume containing 2.5 mM random 9-mers, 1 mM each deoxy-NTP, 8 U placental ribonuclease inhibitor, and the manufacturer’s buffer. Each reaction was allowed to proceed at room temperature for 10–15 min, followed by incubation at 42 C for 90 min.
The K173R genotype was examined in these reverse transcribed samples by PCR using a restriction fragment length polymorphism. PCRs were performed by adding both 2.5 pmol of a sense primer located in exon 1 [5'-GCT CGG GCC CCT AGG AGC-3'; nucleotides (nt) 85–102] and an antisense primer in exon 5 (5'-TGC CAC GAT GCC TGT GTA GTG-3'; nt 853–873) in a 5-µl reaction volume containing a modified buffer (1.5 mM MgCl2 and 5% glycerol) and 0.25 U ExTaq DNA polymerase (Takara Co., Osaka, Japan). Initial denaturation at 96 C for 2 min was followed by 35 cycles of 96 C for 20 sec and 66 C annealing/extension for 30 sec. The PCR-amplified 789-bp fragments were digested overnight with the CvnI restriction enzyme at 37 C, subjected to electrophoresis on a 2% agarose gel, and stained with ethidium bromide. The Lys173 (K) allele was not digested, but the Arg173 (R) allele resulted in two bands of 430 and 359 bp.
To estimate transcriptional efficiency of the 173R or 173K allele, we measured bands intensities in the heterozygotes (i.e. KR genotype), of normal adrenals and APA. For this purpose, we employed the above PCRs for 28–34 cycles (depending on samples) to ensure that the PCRs were in the exponential phase. We repeated the CvnI digestion at least twice to exclude incomplete digestion. Images were loaded into a Macintosh computer with a DC120 digital camera (Eastman Kodak Co., Rochester, NY), and the band intensities were analyzed using Kodak Digital Science 1D Image Analysis software (Eastman Kodak Co.) with øX174-HaeIII fragments as standards. We compared the sum of the 430- and 359-bp band intensities with the 789-bp band intensity in each sample.
Quantitative competitive RT-PCR was used to measure the mRNA levels of CYP11B1 and CYP11B2. The mRNA values were normalized for glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a housekeeping gene, to minimize the effects of variations in deoxyribonuclease digestion and RT between samples. Competitors for each cDNA were prepared following the PCR MIMIC kit protocol (BD Clontech, Palo Alto, CA). Gene-specific primers for each cDNA are as follows: CYP11B1, 5'-TGC GCC TGT TCC TCT ACT CT-3' (sense; nt 1208–1227) and 5'-AGA GAC GTG ATT AGT TGA TG-3' (antisense; nt 1503–1522); CYP11B2, 5'-TAC AGG TTT TCC TCT ACT CG-3' (sense; nt 1208–1227) and 5'-AGA TGC AAG ACT AGT TAA TC-3' (antisense; nt 1503–1522); and GAPDH, 5'-TCA TCA TCT CTG CCC CCT CTG CTG-3' (sense; nt 482–497) and 5'-GAC GCC TGG TTC ACC ACC TTC TTG-3' (antisense; nt 812–832). Competitive PCR was performed by addition of 2.5 pmol of a sense and an antisense primer to 0.5 µl reverse transcribed sample with 0.5 µl of a various range of 2.5 times serially diluted competitor in 5 µl of the above-mentioned PCR buffers, except for no inclusion of glycerol with 0.25 U ExTaq DNA polymerase (TaKaRa Co., Osaka, Japan) for CYP11B1. For CYP11B2, 1.5 mmol/liter MgCl2 was used. Samples were subjected to initial denaturation at 96 C for 2 min, followed by 36–45 cycles of 96 C denaturation for 20 sec, 56 C annealing (GAPDH; 65 C) for 15 sec, and 72 C extension for 30 sec. PCR products were subjected to electrophoresis in 2% agarose gels and stained with ethidium bromide, images were analyzed as described above, and the amount of the competitor whose intensity was equal to the intrinsic template was calculated as the corresponding mRNA level by linear regression. To control for variation due to inclusion of zona fasciculata, especially in normal adrenals, CYP11B2 mRNA levels were also presented as the ratio against CYP11B1 mRNA levels, because the relative ratio of CYP11B2 against CYP11B1 was presumed to differ greatly between zona glomerulosa and zona fasciculata.
Clinical laboratory methods
Peripheral blood samples for hormone levels were drawn from fasting patients in the morning between 0800 and 0900 h in the supine position after resting for at least 30 min. Twenty-four-hour urine samples were collected in plain plastic containers without preservatives. Commercially available RIA kits were used to measure plasma levels of ACTH (ACTH immunoradiometric assay kit, Mitsubishi Petrochemical Co. Ltd., Tokyo, Japan), cortisol (Spac Cortisol kit, Daiichi Radioisotope, Tokyo, Japan), renin activity (Renin RIA Bead, Dainabot RI Laboratory, Tokyo, Japan), or aldosterone and urinary excretion of aldosterone (aldosterone RIA kit, Shionogi Pharmaceutical Co., Osaka, Japan).
Statistical analysis
Results were expressed as the mean ± SD. Mann-Whitney U or Bonferroni/Dunn tests were used for group comparisons. Relationships between two variables were assessed by linear regression. A hypothesis of a gene dosage effect was tested by linear regression analysis with the number of 173R alleles (zero, one, and two, corresponding to KK, KR, and RR genotypes) as the explanatory factor. P < 0.05 was considered significant.
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Results |
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The mRNA levels of CYP11B2 and CYP11B1 in normal adrenals were approximately 0.7% and 20% of the GAPDH mRNA levels, respectively (Table 2). CYP11B2 mRNA levels (B2/G; presented as a ratio against GAPDH) in APA were 34 times higher than those in normal adrenals (P < 0.001), and the mean CYP11B1 mRNA level (B1/G) in APA was 3.9 times higher than in normal adrenals (P = 0.069). Similar results were obtained when CYP11B1 and CYP11B2 expressions were normalized to levels of 18S ribosomal RNA measured by quantitative real-time PCR (not shown). As shown in the lower panels of Fig. 1, spironolactone treatment was associated with higher levels of B1/G (P = 0.004) and B2/G (P = 0.001), but did not affect the B2/B1 ratio. One patient receiving a ß-blocker, who also had liver damage, apparently had high B1/G and B2/G levels. Excluding the data from patients treated with spironolactone or a ß-blocker, B2/G in APA remained 18 times higher than in normal adrenals (Table 2). Subsequent analyses excluded patients receiving spironolactone or a ß-blocker. CYP11B2 mRNA levels relative to CYP11B1 (B2/B1) showed similar patterns (12–13 times higher in APA than in normal adrenals; P = 0.0001). In adenomas causing Cushing’s syndrome, CYP11B2 transcripts could be detected in only four of 17 tissues examined, confirming lower CYP11B2 expression in this type of tumor.
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, lower left), there were no significant correlations between B2/G and plasma aldosterone or urinary excretion of aldosterone (Fig. 2, lower center and right). B2/G also did not correlate with plasma renin activity, serum potassium level, or tumor volume (not shown). B1/G in APA tissues did not significantly correlate with plasma ACTH, but positively correlated with plasma cortisol level (Fig. 2, upper center and right).
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In control adrenals (Fig. 1
, upper panels), the B1/G ratios in each genotype group were 191 ± 104 (x10–3) in KK (n = 6), 151 ± 91 in KR (n = 7), and 262 ± 123 in RR (n = 5). The B2/G ratios were 9.6 ± 6.4 (x10–3) in KK, 8.2 ± 5.3 in KR, and 2.6 ± 1.3 in RR. The B2/B1 ratios were 0.054 ± 0.021 in KK, 0.056 ± 0.013 in KR, and 0.013 ± 0.009 in RR, respectively. B2/G was lower in the RR genotype than in the KK genotype (P = 0.038), and B2/B1 was lower in the RR genotype than in the KK and KR genotypes (P = 0.016 and 0.010).
In patients with APA, there were no differences in serum potassium, plasma renin, or ACTH levels among genotypes (Table 3). The B1/G ratios in each genotype group were 298 ± 225 (x10–3) in KK (n = 11), 457 ± 371 in KR (n = 5), and 300 ± 365 in RR (n = 7). These differences were not significant. However, the B2/G ratios were 171 ± 137 (x10–3) in KK, 158 ± 204 in KR, and 32 ± 26 in RR, thus higher in the KK genotype than in the RR genotype (Fig. 1, lower center; P = 0.043). The B2/B1 ratio was 0.816 ± 0.785 in KK, 0.387 ± 0.263 in KR, and 0.160 ± 0.135 in RR, again higher in the KK genotype than in the RR genotype (Fig. 1, lower right; P = 0.028). Urinary aldosterone excretion was higher in the KK genotype than in the KR genotype, but there were no significant differences between groups in plasma aldosterone, cortisol (Table 3), or tumor volume (not shown).
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Normal adrenals and APA in the KR genotype revealed that transcriptional efficiencies from the 173R allele were 46.6 ± 12.2% (n = 7) and 49.1 ± 20.8% (n = 6), respectively, of those from 173K.
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Discussion |
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Many reports have described the association of the –344T/C polymorphism in CYP11B2 with aldosterone secretion, hypertension, arterial wall stiffness, or cardiac structure (4, 8, 9, 10), but the results have been inconsistent (3). Previously, an association of the –344C allele with hypertension was suggested in Japanese subjects (11), but our larger study did not show any such association (3). To the contrary, several epidemiological studies suggested the association of the –344T allele with hypertension in Scottish (10), French (12), or Japanese (13) populations and with higher blood pressures in Belgian (14), Italian (15), or United Kingdom (16) populations. Moreover, three reports described the association of the –344T allele with higher 24-h urinary excretion of aldosterone (10, 14, 17).
With regard to primary aldosteronism, an Italian group reported the association of a specific haplotype (–344C+[conversion in intron 2]+R173) with idiopathic hyperaldosteronism, but not with APA or essential hypertension (18). The K173 allele, which is always found with the –344T allele, has been proposed to be associated with low-renin essential hypertension in a Chilean population, but this polymorphism does not affect CYP11B2 enzymatic activity in vitro (19). The frequency of the R173 allele in the present 28 APA patients is 0.393, comparable to the frequency of –344C, 0.364 in 535 Japanese normotensives and 0.369 in 360 hypertensives (3). Thus, it is unlikely that CYP11B2 polymorphisms increase the risk of developing an APA.
In this study we attempted to test the hypothesis that CYP11B2 polymorphisms affect adrenal CYP11B2 expression, and indeed, this is the case. We examined the K173R polymorphism instead of –344C/T, because the coding sequence polymorphism, being present in mRNA, could be used to directly compare transcription of the two alleles. CYP11B2 mRNA levels were lowest in samples with the RR genotype of the K173R polymorphism in both normal adrenals and APA. In addition, CYP11B2 mRNA levels varied consistent with a gene dosage effect. Measurement of the band intensities of KR genotype revealed transcription efficiencies from the R173 allele less than half of those from the K173 allele in both normal adrenals and APA. Thus, the R173 allele is correlated with lower CYP11B2 expression in vivo by several methods.
Consistent with this, 24-h urinary aldosterone excretion in APA patients of the KK genotype was higher than in those of the KR genotype. However, there were no significant correlations between adrenal CYP11B2 expression levels and other clinical data, including serum aldosterone and degree of suppression of the main regulators of aldosterone synthesis, renin and potassium (20). Thus, the data suggest a link between genotype and the intermediate phenotype of gene expression, but a less robust correlation of genotype with clinical phenotype.
The –344C promoter allele, which is in complete linkage disequilibrium with the R173 allele, is known to bind the steroidogenic factor-1 (SF-1) transcription factor approximately 4 times more strongly than the –344T allele does. SF-1 is required for normal development of the adrenal gland and for expression of most adrenocortical steroidogenic enzymes (21), which seems inconsistent with the lower CYP11B2 expression of the R173 allele demonstrated in our study. However, SF-1 inhibits human CYP11B2 reporter activity in transfected human adrenocortical cells, in contrast to its effects on all other adrenal steroidogenic enzymes, including CYP11B1 (22), supporting the idea that the –344C allele (and thus the R173 allele) would be associated with lower CYP11B2 expression due to increased binding of SF-1. Considering the opposite effects of SF-1 on CYP11B1 and CYP11B2, B2/B1 ratios might be inversely correlated with intranuclear levels of SF1, but additional studies are necessary to confirm this.
Recently, two studies reported correlations between CYP11B2 polymorphisms and 11ß-hydroxylase activity in vivo (23, 24). These were presumably due to linkage disequilibrium between CYP11B2 and CYP11B1 polymorphisms. In the present study there was no significant correlation of B1/G levels with K173R genotype in either normal adrenals or APA, and so we cannot provide an explanation for the previous observations (23, 24).
In APA, mutations in CYP11B2 itself have not been detected to date. Thus, the mechanism underlying CYP11B2 overexpression might be in transcription-regulating steps. In addition to SF-1, orphan nuclear receptors, such as COUP-TF, DAX-1 (25), or Nurr-1 (26), might be quantitatively or genetically involved. Overproduction of aldosterone in APA might be secondary to cell proliferation rather than dysregulation of its synthesis, so that oncogenes such as p53 gene (27) might be involved. Additional studies will be necessary to clarify these issues.
In conclusion, the present results provide direct evidence that a CYP11B2 haplotype including –344T and K173 is associated with higher gene expression than the –344C/R173 haplotype, favoring reported associations of –344T with higher aldosterone production and blood pressures.
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Footnotes |
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A preliminary presentation of this work was made at the 84th Annual Meeting of The Endocrine Society, San Francisco, CA, June 2002.
First Published Online August 23, 2005
Abbreviations: APA, Aldosterone-producing adenoma; B2/G, CYP11B2/GAPDH ratio; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; nt, nucleotide; SF-1, steroidogenic factor-1.
Received February 11, 2005.
Accepted August 15, 2005.
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References |
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(lower panels), Data from four APA subjects treated with spironolactone; 
, data from an APA patient treated with a ß-blocker; these five subjects were excluded from statistical analysis. Short horizontal lines indicate group mean values. *, P < 0.05.