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Division of Nephrology and Hypertension, Inselspital, University of Berne, 3010 Berne, Switzerland
Address all correspondence and requests for reprints to: Paolo Ferrari, M.D., Department of Nephrology, Fremantle Hospital, University of Western Australia, Alma Street, P.O. Box 480, Fremantle WA 6959 Australia. E-mail: paolo.ferrari{at}health.wa.gov.au.
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Abstract |
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We compared the frequency of two linked CYP11B2 polymorphisms, one in the steroidogenic factor-1 (SF-1) binding site and the other an intronic conversion (Int2) in relation to ARR in 141 hypertensive patients. Patients were divided into groups with either normal or high supine ARR using a cut-off threshold of 145 pmol/liter per ng/liter. Supine ARR was normal in 104 patients and raised in 37 patients. The two polymorphisms were in strong linkage disequilibrium (
2 = 123.8; P < 0.0001). The SF-1 T and Int2 C alleles were more prevalent among patients with high ARR (46% and 43%, respectively) than with normal ARR (22% and 17%; P < 0.01 and P < 0.005, respectively). Odds ratios for raised ARR in subjects with a homozygous SF-1 T and Int2 C haplotype were 6.1 (95% confidence interval, 1.6–22.5; P < 0.005) when compared with the contrasting haplotype. Linear modeling of individual postural changes in renin and aldosterone showed a maximal achievable aldosterone increase of 110 pmol/liter with no mutated haplotype and 500 pmol/liter with two mutated haplotypes. These findings support the view of a molecular basis regulating aldosterone production.
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Introduction |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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The enzyme aldosterone synthase is the key rate-limiting enzyme in the final steps of aldosterone biosynthesis. Angiotensin II is the principal stimulator of aldosterone production (11, 12). Recent reports suggest that variations at the aldosterone synthase gene (CYP11B2) are associated with essential hypertension and may influence aldosterone secretion (13, 14, 15). One of these CYP11B2 polymorphisms (-344 C/T SF-1) is located in the promoter region and influences binding of the transcriptional regulatory protein, SF-1 (16). Another is a gene conversion in intron 2 (Int2 W/C) so that most of the intron has a sequence corresponding to CYP11B1, the gene encoding for the 11ß-hydroxylase, the enzyme involved in the final step of cortisol synthesis (17). These polymorphisms are in linkage disequilibrium (17). The -344 C/T single nucleotide difference at the SF-1 site is thought to alter the sensitivity of CYP11B2 to angiotensin II (14). Thus, inappropriate aldosterone production for the prevailing renin level might be an intermediate phenotypic expression of genetic variation at this locus. Lim et al. (7) showed that more than 90% of hypertensives with an ARR value greater than 750 (pmol/liter per ng/ml/h), to define an abnormally raised ARR, fail to suppress plasma aldosterone with salt loading and fludrocortisone. The relationship between SF-1 genotype, blood pressure, and ARR was analyzed in several studies (18, 19, 20). An increased frequency of the T allele was reported in patients with raised ARR, with the exception of Tamaki et al. (19), who found that homozygosity for the C allele was associated with a higher ARR. The relationship between the Int2 C genotype, blood pressure, and ARR has been analyzed in one single study published recently by Lim et al. (21), who found an increased prevalence of Int2 C in patients with ARR greater than 1000 pmol/liter/ng/ml/h. These authors also showed a significant excess of the T allele of SF-1 and the Int2 C allele in patients with a raised ARR (21).
We therefore investigated the distribution of the two linked polymorphic loci (SF-1 and Int2) at the CYP11B2 gene in two groups of hypertensive patients stratified in relation to their ARR. Study design and ARR cut-off were similar to those chosen by Lim et al. (21). In contrast, the present study characterized the ARR with plasma immunoreactive renin and analyzed the functional role of the investigated genotypes in relation to the postural changes in plasma renin and aldosterone levels.
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Patients and Methods |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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Between January 2000 and December 2001, we investigated 141 consecutive patients referred for further assessment to our specialist hypertension clinic in a tertiary care university teaching hospital. In most cases, the reason for the assessment was the presence of resistant hypertension or the requirement of three or more antihypertensives to control blood pressure. All had raised office blood pressure (BP) readings (systolic BP > 140 mm Hg and/or diastolic BP > 90 mm Hg) under therapy confirmed over a period of at least 3 months before their referral by their physicians. Patients who were referred by their primary care physicians for the management of presumed pheochromocytoma or primary aldosteronism, because of hormonal values suggesting these conditions, and hypertensive patients with a known adrenal mass or on spironolactone treatment were excluded. Also, patients with renal diseases, renovascular hypertension, Cushing’s syndrome, obstructive sleep apnea, thyroid disease, and hyperparathyroidism were excluded.
Antihypertensive medication was stopped for 7–10 d in all patients to allow the influence of medication on endocrine function to be minimized. To control hypertension when needed, the calcium channel blocker amlodipine (10 mg/d) with or without the 
-blocker prazosin (up to 10 mg/d) were administered; both medications are least likely to affect measurements of renin and aldosterone (22, 23).
Hormonal assays
After an overnight fast, blood samples were taken for plasma immunoreactive renin (irR; nanograms per liter) and aldosterone (picomoles per liter) after the patients had been in the supine position for 1 h and again after 1 h in the upright position. ARR was derived dividing plasma aldosterone by plasma irR. We chose an ARR of 145 pmol/liter per ng/liter as a cut-off to categorize our patients (3, 24). This value corresponds to an ARR of 750 pmol/liter per ng/ml/h, which was found to be highly predictive of primary aldosteronism (7). irR was measured by the Bio-Rad Renin III immunoradiometric assay (Bio-Rad Laboratories, Inc., Hertfordshire, UK) with an intraassay coefficient of variation of less than 12% between 3 and 320 ng/liter. Plasma aldosterone was measured by a solid-phase (coated) RIA technique, PCP Coat-A-Count assay (Diagnostic Products, Los Angeles, CA), with an intraassay coefficient of variation of less than 11% between 70 and 3300 pmol/liter.
Genotyping
Genomic DNA was isolated from peripheral blood, and detection of polymorphisms was performed by PCR analysis as previously described (25). Polymorphism detection for the SF-1 polymorphic marker of the CYP11B2 genes was performed with the primers and conditions using established protocols from our laboratory (25). The 228-bp amplicon contains two HaeIII restriction enzyme sites (GGCC). The presence of a C to T transition at position -344 (GGCT) removes one of these sites. After digestion, individuals homozygous for the transition (TT) produce two bands of 175 and 53 bp; individuals homozygous for the wild type (CC) produce three bands of 104, 71, and 53 bp; and heterozygous individuals (TC) produce four bands. The Int2 C genotype was analyzed by use of two separate PCRs, one that amplifies the normal gene and one that amplifies the Int2 C (17), with the primers reported previously by Davies et al. (14). The size of the amplicon in each reaction is approximately 418 bp.
Functional genomic analysis
In unselected normotensive and hypertensive subjects, significant increments of plasma renin occur rapidly upon rising from the supine to the upright position (26, 27). The increments of plasma aldosterone are initially delayed, but peak levels are achieved approximately at the same time as peak renin levels between 60 and 90 min (26, 27). To analyze whether the CYP11B2 genotype affects postural response of plasma renin and aldosterone, postural changes in renin (
Renin = upright-supine irR) and aldosterone (Aldosterone = upright-supine aldosterone) were assessed in relation to the genotype. The ratio of the postural changes in the aldosterone to renin was calculated by dividing Aldosterone by Renin. A linear fit of individual Renin/Aldosterone vs. Renin was plotted for identified genotypes. The relationship between the two parameters appears to fit a Michaelis-Menten type equation. Thus, the maximal achievable aldosterone increase (Aldomax) and Km values were calculated from the slope and intercept of the plot, obtained with the linearized Michaelis-Menten equation: Renin/Aldosterone = 1/Aldomax x Renin + Km/Aldomax.
Statistical analysis
Statistical differences between means were assessed by t test or ANOVA for analysis of continuous variables and by nonparametric analysis using the Wilcoxon or Kruskal-Wallis test for variables that were not normally distributed. Odds ratios and confidence intervals were calculated in the standard way, with log transformations and a normal approximation being used for the confidence intervals. For comparisons of genotype distributions in different groups, the 2 test was used, taking an ARR threshold of 145. Comparisons for the number of mutated haplotypes were performed for three groups: no mutated haplotypes (SF-1 CC, Int2 WW), two mutated haplotypes (SF-1 TT, Int2 CC) and one mutated and one nonmutated haplotype (SF-1 CT, Int2 WC). Values are expressed as mean ± SD. All statistical analyses were performed using the Systat 10 (SPSS, Inc., Chicago, IL) statistical software package.
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Results |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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Basic demographic and biochemical data of the 141 patients, grouped according to the supine ARR cut-off level of 145 pmol/liter per ng/liter are reported in Table 1
. All patients had both ARR and genetic data. In 78 patients, hormonal analysis was performed without antihypertensive drug therapy, whereas the others received amlodipine (n = 47) or amlodipine plus prazosin (n = 16). The distribution of patients with or without antihypertensive agents during hormonal testing did not differ among the 104 (74%) patients with supine ARR of 145 or less and the 37 (26%) patients with supine ARR greater than 145. Patients with an increased supine ARR had significantly higher plasma sodium and bicarbonate and lower plasma potassium values than subjects with normal ARR (Table 1).
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). In subjects with raised supine ARR, the ratio of the postural changes in aldosterone to changes in renin was larger than in subjects with normal supine ARR (F-ratio, 11.03; P < 0.001). The number of patients with raised ARR in both the supine and upright positions was 32 (23%), whereas five patients with raised supine ARR had normal upright ARR and four patients with normal supine ARR had raised upright ARR.
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Genotype and haplotype analysis
The CYP11B2 genotype distribution within the study population is shown in Table 2. Each of the polymorphisms was in Hardy-Weinberg equilibrium. However, the SF-1 and intron 2 polymorphisms were in strong linkage disequilibrium (2 = 123.8; P < 0.0001). The -344C allele was completely linked with a normal intron 2. Table 3 summarizes the SF-1 and Int2 distributions with respect to ARR. Using a supine ARR threshold of 145, patients with a raised ratio had a statistically significant excess of the T allele at the SF-1 site and of the conversion polymorphism at the Int2 site.
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. In this study population, 32 subjects (23%) had no mutated haplotype, 44 (31%) had one mutated haplotype, and 28 (20%) had two mutated haplotypes. There was a significant association between ARR greater than 145 and the number of these haplotypes (Fig. 2). Among patients with ARR greater than 145, there were 35% of subjects with two mutated haplotypes compared with 22% with one mutated haplotype or 11% with the contrasting haplotype. Thus, an ARR greater than 145 was more likely to occur when patients had two mutated haplotypes (SF-1 TT, Int2 CC) compared with one [odds ratio, 3.9; 95% confidence interval (CI), 1.3–11.5; P < 0.01)] or no mutated haplotypes (odds ratio, 6.1; 95% CI, 1.6–22.5; P < 0.005).
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Functional genomic analysis
The postural increase in plasma renin (Renin) tended to be more marked in patients with the CC than with the TT genotype (+9.4 ± 16.2 vs. +3.7 ± 8.6 ng/liter, SF-1 CC vs. TT; P = 0.05), and similarly with no mutated than with two mutated haplotypes (+9.4 ± 16.2 vs. +4.2 ± 7.8 ng/liter, haplotypes 0 vs. 2; P < 0.05). The increment in aldosterone (Aldosterone) upon standing was similar in the two groups for the SF-1 genotype (+170 ± 215 vs. +200 ± 173 pmol/liter) or haplotype analysis (+170 ± 215 vs. +204 ± 174 pmol/liter). The postural Aldosterone to Renin ratio was significantly smaller in subjects with the CC than the TT genotype (F-ratio, 4.63; P = 0.011) or with no mutated vs. two mutated haplotypes (F-ratio, 4.01; P = 0.021; Fig. 3), but this relationship was weaker with intron 2 alone (F-ratio, 2.826; P = 0.05).
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2 = 14.5, P < 0.001). In these patients the odds ratios for two mutated haplotypes vs. no mutated haplotype for an ARR greater than 145 in both the supine and upright position was 13.0 (95% CI, 2.51–67.42; P < 0.0001).
When the linear fit of individual Renin/Aldosterone vs. Renin was calculated for the identified genotypes, the Aldomax was 110 pmol/liter with no mutated haplotype, 435 pmol/liter with one mutated haplotype, and 500 pmol/liter with two mutated haplotypes.
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Discussion |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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Previous studies suggested that the activity of the aldosterone synthase may be genetically determined. Davies et al. (14) demonstrated that subjects with the SF-1 T allele have a higher urinary aldosterone excretion rate than those with the C allele, and the SF-1 T allele was reported to be more common in hypertensive than in normotensive subjects (14, 28). Some (18, 20), but not all (19) studies that analyzed the relationship between SF-1 genotype, blood pressure, and ARR reported an increased frequency of the SF-1 T allele in patients with raised ARR as opposed with low ARR. One contrasting finding in a Japanese population (19) might be either a consequence of a genetic difference between populations or a result of a selection bias, because the frequency of the T allele in those patients was 0.74 compared with 0.53–0.56 in Western populations (14, 28, 29). One large study did not find any significant association between SF-1 polymorphism of CYP11B2 and variability of serum aldosterone levels, blood pressure, or cardiac size or function (30). In that cohort of the MONICA study, some of the patients were on antihypertensive drugs (30), a confounding factor that substantially compromises the interpretation of renin and aldosterone levels (3).
So far, the relationship between the Int2 C genotype, blood pressure, and ARR has been analyzed in one study published recently by Lim et al. (21). These authors found an increased prevalence of Int2 C in patients with ARR greater than 1000 pmol/liter/ng/ml/h, and showed significant excesses of the T allele of SF-1 and the Int2 C allele in patients with a raised ARR (21). Moreover, haplotype analysis revealed that the highest odds ratio for an increased ARR was in subjects homozygous for SF-1 T and Int2 C alleles (21), an observation consistent with our findings. It has been shown that both bias and genuine population diversity might explain why early association studies tend to overestimate the disease protection or predisposition conferred by a genetic polymorphism (31). The present findings, together with the observation by Lim et al. (21) in two independent populations strongly support the notion that molecular variants of CYP11B2 are indeed functionally relevant and result in an intermediate phenotype of increased ARR and hypertension as the distant phenotype. The higher increase in the postural changes in aldosterone vs. renin found in our hypertensives with the TT genotype is in line with the observation of Rossi et al. (20) of an increased postcaptopril ARR with TT genotype than with CC genotype in low renin hypertension (18, 20). The two stimuli for renin secretion are nevertheless not similar in their relation to aldosterone secretion, because with captopril, angiotensin II is expected to drop. Although it is not known whether captopril treatment in these patients would suppress angiotensin II generation to the same extent as controls, high postcaptopril aldosterone levels for the prevailing circulating renin could in part suggest angiotensin II-independent aldosterone production.
Despite the strong association between genotype and raised ARR found in our study, the differences in prevalence of the SF-1 T and Int2 C alleles in the two groups were not sufficient to discriminate subjects with raised and low ARR. The possibility of diurnal variation in aldosterone secretion independent from renin and angiotensin II was minimized by sampling the blood for hormonal assay after an overnight fast in the morning between 0800 and 0900 h. Another reason could be that a single ARR may lead to an overestimation of the true prevalence of abnormal aldosterone regulation. This seems to be the case, because of the 37 patients with raised supine ARR, only 32 also had increased ARR in the upright position. When the genotype was analyzed in patients with both raised supine and upright ARR compared with subjects with normal ARR in either the supine and/or upright position, then the CC genotype was predictive of a normal ARR in 94% of the patients. It has been suggested that the natural history of hypertension proceeds from essential (high to normal renin) hypertension through to low renin hypertension to idiopathic aldosteronism over time, a condition that has been described as tertiary aldosteronism (1). The rate of this progression may be different, depending on genetic susceptibility (21). Thus, it is possible that polymorphisms of CYP11B2 may be predictive for abnormal aldosterone production in older subjects only.
In the hypertensive patients referred for further assessment to our specialist hypertension clinic, the prevalence of aldosteronomas was 4.2%. This percentage is in line with previous reports (3). All of these patients had increased ARR in excess of 400 pmol/ng and plasma aldosterone levels greater than 500 pmol/liter. However, although five of the six patients had one or two mutated haplotypes, the CYP11B2 genotype among patients with aldosteronoma was not unique, arguing against a clonal selection through aldosterone overproduction because of the SF-1/Int2 genotype as a mechanism for the development of aldosterone-producing adenomas.
The exact mechanism for increased aldosterone synthase activity with the T allele in the CYP11B2 promoter is still the object of debate (17, 32). The ratio of the change in plasma aldosterone to the change in plasma renin was significantly smaller in subjects with the CC than the TT genotype or with no mutated vs. two mutated haplotypes. This suggests that with smaller increases in circulating renin, and thus angiotensin II, the increases in plasma aldosterone are more substantial. Because there is little evidence for an increased hyperresponsiveness of angiotensin II to renin in these patients, these findings point to an increased sensitivity to angiotensin II in the presence of the TT genotype in the promoter of CYP11B2, as previously hypothesized (14). Alternative explanations that need consideration are increased transcription factor availability at other functional sites of the gene, the possibility of transcriptional activation of the steroidogenic acute regulatory gene by a SF-1-dependent mechanism (33) or linkage of the SF-1 and Int2 C sites with a quantitative trait locus in the regulatory elements. The fact that renin levels are not suppressed and stimulate with postures could suggest an abnormal regulation of renin in addition to the abnormal aldosterone regulation. This is, however, unlikely because some patients with aldosteronomas are also responsive to small increases in the plasma level of angiotensin II and have a normal plasma aldosterone response on postural testing (34).
In conclusion, these findings support the view that regulation of aldosterone production has a strong genetic component. A single ARR may lead to an overestimation of the true prevalence of abnormal aldosterone regulation; however, the CC genotype is predictive of a normal ARR in 94% of the selected patients with normal supine and upright ARR. Whether these findings apply to unselected hypertensive patients remains to be demonstrated.
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Footnotes |
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Abbreviations: Aldomax, Maximal achievable aldosterone increase; ARR, aldosterone to renin ratio; CI, confidence interval; Int2, intronic conversion; irR, immunoreactive renin; SF-1, steroidogenic factor-1.
Received October 16, 2002.
Accepted March 7, 2003.
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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145 (dashed line) or >145 (solid line). Results are mean ± SEM. The line from the origin indicates the ARR cut-off.
