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The Journal of Clinical Endocrinology & Metabolism 88(6):2495–2500
Copyright © 2003 by The Endocrine Society
doi: 10.1210/jc.2002-021598
A Biallelic Gene Polymorphism of CYP11B2 Predicts
Increased Aldosterone to Renin Ratio in Selected
Hypertensive Patients
JÉRÔME NICOD, DAVID BRUHIN, LUCAS AUER, BRUNO VOGT, FELIX J. FREY,
PAOLO FERRARI
AND
Division of Nephrology and Hypertension, Inselspital, University of Berne, 3010 Berne, Switzerland
Altered control of aldosterone synthase (CYP11B2) gene expression may modulate aldosterone secretion, as suggested by
a raised aldosterone to renin ratio (ARR) in some patients
with essential hypertension.
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 ⴝ 123.8;
P < 0.0001). The SF-1 T and Int2 C alleles were more prevalent
I
N RECENT YEARS, the role of a primary activation of the
aldosterone axis in the pathogenesis of essential hypertension has attracted some interest (1). It has been estimated
that approximately one third of the hypertensive population
has low renin levels, with a higher proportion of low renin
in blacks than in whites (2). Moreover, up to 15% of unselected hypertensives have a raised aldosterone to renin
ratio (ARR) in plasma (3), and in most of these subjects
plasma aldosterone is only partially suppressible on salt
loading, the current diagnostic criterion for primary aldosteronism (4 –7). Some authors support the view that this
finding indicates a high prevalence of primary aldosteronism
among patients with hypertension (8), although this opinion
is not shared by others (9). Nevertheless, measurements of
the ARR are also valuable, because they are predictive of the
blood pressure response to spironolactone treatment (10).
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 (13–15). 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 11␤-hydroxylase, the enzyme in-
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.
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. (J Clin Endocrinol Metab
88: 2495–2500, 2003)
volved 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 –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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J Clin Endocrinol Metab, June 2003, 88(6):2495–2500
Patients and Methods
Patients
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).
Nicod et al. • CYP11B2 and ARR
⌬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 ⫻ ⌬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.
Results
Hormonal assays
General characteristics
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.
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).
In patients with raised ARR compared with normal supine
ARR, the average postural increase in plasma irR was smaller
(⫹0.9 ⫾ 0.8 vs. ⫹6.2 ⫾ 17 ng/liter; P ⬍ 0.005), whereas the
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 ⫽ uprightsupine 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.
TABLE 1. Baseline demographic and biochemical data according
to the supine ARR group (mean ⫾ SD)
ARR
No.
Age (yr)
Gender (M/F)
Body mass index (kg/m2)
Blood pressure (mm Hg)
Heart rate (bpm)
Plasma
Sodium (mmol/liter)
Potassium (mmol/liter)
Bicarbonate (mmol/liter)
Uric acid (␮mol/liter)
Creatinine (␮mol/liter)
Urine
Sodium (mmol/24 h)
Potassium (mmol/24 h)
ⱕ145
⬎145
104
50 ⫾ 14
51/53
26.8 ⫾ 4.8
165/100 ⫾ 27/17
73 ⫾ 11
37
51 ⫾ 13
17/20
28.8 ⫾ 6.6
168/103 ⫾ 30/18
70 ⫾ 16
140 ⫾ 2
3.9 ⫾ 0.4
24.5 ⫾ 2.3
356 ⫾ 92
102 ⫾ 22
142 ⫾ 2a
3.7 ⫾ 0.4a
27.4 ⫾ 2.9a
332 ⫾ 81a
102 ⫾ 18
189 ⫾ 85
67 ⫾ 31
194 ⫾ 78
73 ⫾ 27
M, Male; F, female; bpm, beats per minute.
a
P ⬍ 0.05 high vs. normal group.
Nicod et al. • CYP11B2 and ARR
mean postural increase in plasma aldosterone was comparable (⫹179 ⫾ 125 vs. ⫹197 ⫾ 194 pmol/liter; P ⫽ not significant; Fig. 1). 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.
In 13 subjects, supine plasma aldosterone concentration
was greater than 500 (781 ⫾ 213) pmol/liter and the ARR was
greater than 145 (460 ⫾ 235). Among those patients, six were
found to have supine plasma aldosterone greater than 800
(964 ⫾ 142) pmol/liter. A unilateral adrenal mass was demonstrated in all of them by magnetic resonance imaging. In
the six patients with adenoma, postural changes of aldosterone were on average 34 ⫾ 28% and less than 80% in all cases,
and ARR was increased in both the supine and upright
positions. Hypertension was cured by surgical removal of the
tumor in all subjects. When these patients were excluded
from the analysis, comparisons between groups with normal
or raised ARR remained comparable to the results in the 141
patients selected.
J Clin Endocrinol Metab, June 2003, 88(6):2495–2500 2497
TABLE 2. Distribution of the SF-1 and intron 2 genotypes
of CYP11B2
SF-1
Intron 2
WW
WC
CC
CC
TC
TT
32
0
0
19
44
6
3
9
28
For intron 2: W, wild-type; C, conversion.
TABLE 3. Genotype distribution by supine ARR groups
Genotype
SF-1
CC
CT
TT
Intron 2
WW
WC
CC
ARR
ⱕ145
⬎145
0.88
0.77
0.57
0.12
0.23
0.43
0.83
0.77
0.53
0.17
0.23
0.47
␹2
P
8.9
0.012
10.5
0.005
For intron 2: W, wild-type; C, conversion.
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.
The relationship between supine ARR and the number of
mutated haplotypes (SF-1 T and Int2 C alleles) associated
with a raised ARR is shown in Fig. 2. In this study population,
32 subjects (23%) had no mutated haplotype, 44 (31%) had
FIG. 1. Relationship between supine (S) and upright (U) plasma irR
and plasma aldosterone in hypertensive patients with ARRs ⱕ 145
(dashed line) or ⬎145 (solid line). Results are mean ⫾ SEM. The line
from the origin indicates the ARR cut-off.
FIG. 2. Percentage of patients by mutated SF-1 T/Int2 C haplotypes
of CYP11B2 according to the supine ARR. Black bars are patients
with ARR ⬎ 145 (n ⫽ 23); white bars are patients with ARR ⱕ 145
(n ⫽ 82). Difference between ARR groups, ␹2 value 10.84; P ⬍ 0.005.
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).
Of the six patients with adenoma, three had two mutated
haplotypes, two had one mutated haplotype, and one had the
contrasting haplotype. In the one patient without mutated
haplotypes, baseline biochemical values indicated mineralocorticoid hypertension (plasma potassium, 2.4 mmol/liter;
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J Clin Endocrinol Metab, June 2003, 88(6):2495–2500
plasma sodium, 147 mmol/liter; bicarbonate, 34 mmol/liter;
and uric acid, 239 ␮mol/liter)
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).
Genotype frequencies were compared for the 32 patients
with raised ARR in both the supine and upright positions vs.
the 109 subjects with a normal ARR in the supine and/or
upright position. In patients with raised ARR, the genotype
distribution for the SF-1 was 0.43 for the TT and 0.06 for the
CC alleles, whereas in patients with normal ARR it was 0.57
for the TT and 0.94 for the CC alleles (␹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
FIG. 3. Postural changes in the ARR according to SF-1 genotype (top)
and number of mutated haplotypes of CYP11B2 (bottom). Significance
values are by ANOVA with Bonferroni adjustment. Results are
mean ⫾ SEM.
Nicod et al. • CYP11B2 and ARR
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.
Discussion
The findings of the present study indicate that the prevalence of SF-1 T and Int2 C alleles of the aldosterone synthase
(CYP11B2) gene is significantly higher in selected hypertensive subjects with an increased ARR than in patients with a
low ARR. An association between raised ARR and the number of SF-1 T/Int2 C haplotypes is also observed. When
postural aldosterone and renin changes are analyzed, the
Aldomax is higher in subjects with two haplotypes as compared with subjects with no mutated haplotypes. These observations support the view that if variations in CYP11B2
gene contribute to hypertension, this may result in an intermediate phenotype with apparent aldosterone excess relative to the prevailing renin levels (14, 20, 21).
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 sup-
Nicod et al. • CYP11B2 and ARR
port 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 angio-
J Clin Endocrinol Metab, June 2003, 88(6):2495–2500 2499
tensin 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.
Acknowledgments
Received October 16, 2002. Accepted March 7, 2003.
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: [email protected].
This work was supported in part by a grant from the Swiss National
Research Foundation (3100-58889) and the Cloëtta Foundation, Zurich,
Switzerland.
References
1. Lim PO, Struthers AD, MacDonald TM 2002 The neurohormonal natural
history of essential hypertension: towards primary or tertiary aldosteronism?
J Hypertens 20:11–15
2. Brunner HR, Laragh JH, Baer L, Newton MA, Goodwin FT, Krakoff LR, Bard
RH, Buhler FR 1972 Essential hypertension: renin and aldosterone, heart
attack and stroke. N Engl J Med 286:441– 449
3. Ferrari P, Bonny O 2003 Forms of mineralocorticoid hypertension. Vitam
Horm 66:113–156
4. Luetscher JA, Weinberger MH, Dowdy AJ, Nokes GW, Balikian H, Brodie
A, Willoughby S 1969 Effects of sodium loading, sodium depletion and posture on plasma aldosterone concentration and renin activity in hypertensive
patients. J Clin Endocrinol Metab 29:1310 –1318
5. Coghlan JP, Doyle AE, Jerums G, Scoggins BA 1972 The effects of sodium
loading and deprivation on plasma renin and plasma and urinary aldosterone
in hypertension. Clin Sci 42:15–23
6. Gordon RD 1995 Primary aldosteronism. J Endocrinol Invest 18:495–511
7. Lim PO, Dow E, Brennan G, Jung RT, MacDonald TM 2000 High prevalence
of primary aldosteronism in the Tayside hypertension clinic population. J Hum
Hypertens 14:311–315
8. Stowasser M 2001 Primary aldosteronism: revival of a syndrome. J Hypertens
19:363–366
9. Kaplan NM 2001 Cautions over the current epidemic of primary aldosteronism. Lancet 357:953–954
10. Lim PO, Jung RT, MacDonald TM 1999 Raised aldosterone to renin ratio
predicts antihypertensive efficacy of spironolactone: a prospective cohort
follow-up study. Br J Clin Pharmacol 48:756 –760
11. Tobian L 1967 Renin release and its role in renal function and the control of
salt balance and arterial pressure. Fed Proc 26:48 –54
2500 J Clin Endocrinol Metab, June 2003, 88(6):2495–2500
12. Miller RE, Vander AJ, Kowalczyk S, Geelhoed GW 1968 Aldosterone secretion and plasma renin during renin infusion and acute salt depletion. Am J
Physiol 214:228 –231
13. Takeda Y, Furukawa K, Inaba S, Miyamori I, Mabuchi H 1999 Genetic
analysis of aldosterone synthase in patients with idiopathic hyperaldosteronism. J Clin Endocrinol Metab 84:1633–1637
14. Davies E, Holloway CD, Ingram MC, Inglis GC, Friel EC, Morrison C,
Anderson NH, Fraser R, Connell JM 1999 Aldosterone excretion rate and
blood pressure in essential hypertension are related to polymorphic differences in the aldosterone synthase gene CYP11B2. Hypertension 33:703–707
15. Paillard F, Chansel D, Brand E, Benetos A, Thomas F, Czekalski S, Ardaillou
R, Soubrier F 1999 Genotype-phenotype relationships for the renin-angiotensinaldosterone system in a normal population. Hypertension 34:423– 429
16. Bassett MH, Zhang Y, Clyne C, White PC, Rainey WE 2002 Differential
regulation of aldosterone synthase and 11␤-hydroxylase transcription by steroidogenic factor-1. J Mol Endocrinol 28:125–135
17. White PC, Slutsker L 1995 Haplotype analysis of CYP11B2. Endocr Res 21:
437– 442
18. Komiya I, Yamada T, Takara M, Asawa T, Shimabukuro M, Nishimori T,
Takasu N 2000 Lys(173)Arg and ⫺344T/C variants of CYP11B2 in Japanese
patients with low-renin hypertension. Hypertension 35:699 –703
19. Tamaki S, Iwai N, Tsujita Y, Kinoshita M 1999 Genetic polymorphism of
CYP11B2 gene and hypertension in Japanese. Hypertension 33:266 –270
20. Rossi E, Regolisti G, Perazzoli F, Negro A, Davoli S, Nicoli D, Sani C, Casali
B 2001 ⫺344C/T polymorphism of CYP11B2 gene in Italian patients with
idiopathic low renin hypertension. Am J Hypertens 14:934 –941
21. Lim PO, Macdonald TM, Holloway C, Friel E, Anderson NH, Dow E, Jung
RT, Davies E, Fraser R, Connell JM 2002 Variation at the aldosterone synthase
(CYP11B2) locus contributes to hypertension in subjects with a raised aldosterone-to-renin ratio. J Clin Endocrinol Metab 87:4398 – 4402
22. Barbieri C, Ferrari C, Caldara R, Rampini P, Crossignani RM, Bergonzi M
1981 Effects of chronic prazosin treatment on the renin-angiotensin-aldosterone system in man. J Clin Pharmacol 21:418 – 423
23. Carpene G, Rocco S, Opocher G, Mantero F 1989 Acute and chronic effect of
nifedipine in primary aldosteronism. Clin Exp Hypertens A 11:1263–1272
Nicod et al. • CYP11B2 and ARR
24. Trenkel S, Seifarth C, Schobel H, Hahn EG, Hensen J 2002 Ratio of serum
aldosterone to plasma renin concentration in essential hypertension and primary aldosteronism. Exp Clin Endocrinol Diabetes 110:80 – 85
25. Lovati E, Richard A, Frey BM, Frey FJ, Ferrari P 2001 Genetic polymorphisms
of the renin-angiotensin-aldosterone system in end-stage renal disease. Kidney
Int 60:46 –54
26. Tuck ML, Dluhy RG, Williams GH 1975 Sequential responses of the reninangiotensin-aldosterone axis to acute postural change: effect of dietary sodium.
J Lab Clin Med 86:754 –763
27. Weidmann P, Beretta-Piccoli C, Ziegler WH, Keusch G, Gluck Z, Reubi FC
1978 Age versus urinary sodium for judging renin, aldosterone, and catecholamine levels: studies in normal subjects and patients with essential hypertension. Kidney Int 14:619 – 628
28. Brand E, Chatelain N, Mulatero P, Fery I, Curnow K, Jeunemaitre X, Corvol
P, Pascoe L, Soubrier F 1998 Structural analysis and evaluation of the aldosterone synthase gene in hypertension. Hypertension 32:198 –204
29. Pojoga L, Gautier S, Blanc H, Guyene TT, Poirier O, Cambien F, Benetos A
1998 Genetic determination of plasma aldosterone levels in essential hypertension. Am J Hypertens 11:856 – 860
30. Schunkert H, Hengstenberg C, Holmer SR, Broeckel U, Luchner A, Muscholl
MW, Kurzinger S, Doring A, Hense HW, Riegger GA 1999 Lack of association
between a polymorphism of the aldosterone synthase gene and left ventricular
structure. Circulation 99:2255–2260
31. Ioannidis JP, Ntzani EE, Trikalinos TA, Contopoulos-Ioannidis DG 2001
Replication validity of genetic association studies. Nat Genet 29:306 –309
32. Clyne CD, Zhang Y, Slutsker L, Mathis JM, White PC, Rainey WE 1997
Angiotensin II and potassium regulate human CYP11B2 transcription through
common cis-elements. Mol Endocrinol 11:638 – 649
33. Clark BJ, Combs R 1999 Angiotensin II and cyclic adenosine 3⬘, 5⬘-monophosphate induce human steroidogenic acute regulatory protein transcription
through a common steroidogenic factor-1 element. Endocrinology 140:4390 –
4398
34. Irony I, Kater CE, Biglieri EG, Shackleton CH 1990 Correctable subsets of
primary aldosteronism. Primary adrenal hyperplasia and renin responsive
adenoma. Am J Hypertens 3:576 –582