(Hypertension. 1999;33:703-707.)
© 1999 American Heart Association, Inc.
Scientific Contribution |
From the MRC Blood Pressure Group (E.D., C.D.H., M.C.I., G.C.I., R.F., J.M.C.C.), Department of Medicine and Therapeutics (E.C.F., N.H.A.), Western Infirmary, Glasgow, Scotland; and the MONICA Project (C.M.), Glasgow Royal Infirmary, Glasgow, Scotland.
Correspondence to Dr Robert Fraser, MRC Blood Pressure Group, Western Infirmary, Glasgow G11 6NT, Scotland.
| Abstract |
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Key Words: : polymorphism binding sites, SF-1 intronic conversion blood pressure aldosterone
| Introduction |
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Expression of the gene-encoding aldosterone synthase (CYP11B2) is regulated by Ang II and potassium12 ; a chimeric rearrangement of this gene with the adjacent gene-encoding 11ß-hydroxylase (CYP11B1) is known to result in GSH.13 This locus is, therefore, an important candidate region in other forms of hypertension.
Steroidogenic enzyme gene promoter regions contain sites for interaction with a variety of control factors, one of which is steroidogenic factor-1 (SF-1).14 15 Differences in the structure of this site may also alter sensitivity to Ang II. A polymorphism at this site has been described that is reported to be associated with altered left ventricular mass in a Finnish population.16 Further, White and Slutsker17 have recently described another polymorphism in which intron 2 of CYP11B1 has been transferred to CYP11B2. It is not yet known whether this affects expression of the gene or stability of mRNA, and there are no reports of the influence of this polymorphism in cardiovascular disease. In the present study, the distribution of the SF-1 binding site and intron conversion (IC) polymorphisms and their relationship to aldosterone excretion rate have been compared in large groups of hypertensive and normotensive subjects.
| Methods |
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Control Subjects
Control subjects were drawn from the North Glasgow
coronary risk survey that had about 200 randomly selected
members of the North Glasgow population in each 10-year age/sex band
from 25 to 64 years. They were normotensive (<140/90), and none were
receiving antihypertensive therapy, treatment for heart disease, or
hormone replacement therapy. They were individually age- and
sex-matched with the cases by random selection from all controls that
matched the criteria of the cases. BP was measured on 2 occasions by
use of the Hawksley random zero sphygmomanometer. The results were
averaged.
Population Survey of Urinary Tetrahydroaldosterone
Metabolite Excretion
A larger sample (n=486) of the MONICA IV survey community
subjects agreed to perform a 24-hour urine collection for assay of
tetrahydroaldosterone (THaldo).
Laboratory Methods
Genotyping
Blood for genotyping was taken into EDTA-containing receptacles;
DNA was extracted by use of a standard phenol-chloroform method and
stored at -20°C until required for batch genotyping.
The region of DNA containing the HaeIII/SF-1 polymorphism was amplified by PCR by use of conditions similar to those previously described. The primers used are shown in Table 1. A PCR product of 228 bp was amplified. This was digested with HaeIII and subjected to electrophoresis in 3% MetaPhor agarose.
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The 228 bp amplicon contains 2 HaeIII restriction enzyme sites (GG CC). The presence of a C to T transition at position -344 (GG CT) removes one of these sites. After digestion, individuals homozygous for the transition (TT) produce 2 bands of 175 and 53 bp, individuals homozygous for the wild-type (CC) produce 3 bands of 104, 71, and 53 bp, and heterozygous individuals (TC) produce 4 bands.
The IC was genotyped by use of 2 separate PCR reactions, one that amplifies the normal gene (WT) and one that amplifies the conversion.17 The 2 pairs of primers and the conditions used are shown in Table 1. The size of the amplicon in each reaction is approximately 418 bp.
Urinary Corticosteroid Metabolite
Measurements
Urine samples were collected for a 24-hour period in plain
plastic containers without preservative. Aliquots of urine were stored
at -22°C until required for THaldo assay by gas
chromatography/mass spectroscopy by use of the method
of Shackleton18 with minor modifications.
Statistical Methods
Comparisons between cases and controls of demographic
variables and genotype or allele frequencies were
carried out by paired t tests and McNemar's test,
respectively. In particular, a variation of McNemar's test appropriate
for case-control comparisons involving either 2x2 or 3x3 contingency
tables was used with analyses by allele or
genotype, respectively.19 Hardy-Weinberg
equilibrium was checked by a
2 test, and the
strength of allelic association between SF-1 and IC polymorphisms
was estimated by Jurg Ott's EH program.20 Median THaldo
levels were compared between genotype groups by use of a
Kruskal-Wallis test.
| Results |
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Genetic Analysis
The general population frequency of the 2 polymorphisms was
first studied in a random selection of the normal population from the
MONICA IV community survey (n=256). The SF-1 binding-site
polymorphism showed population frequencies of 0.49 (C allele)
and 0.51 (T allele). For the intron 2 polymorphism, the
wild-type allele had a frequency of 0.48 and the conversion
allele a frequency of 0.52. There was significant linkage between
the 2 polymorphisms so that the 3 common haplotypes were observed:
C/wild-type (0.45), T/wild-type (0.13), and T/conversion (0.38).
The distribution of genotypes and alleles for the 2 polymorphisms in the case-control populations is shown in Table 3. The control group was at Hardy-Weinberg equilibrium for both polymorphisms, whereas the case group was in Hardy-Weinberg equilibrium for the IC polymorphism but not for the SF-1 binding site (P=0.0007).
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There was a relative excess of TT homozygotes for the SF-1 binding site in the cases compared with controls (P=0.042), and a similar excess of T alleles (or deficiency of C alleles) was noted when analysis was done by allele counting (P=0.009). Similarly, for the IC, there was an excess of the conversion allele (P=0.016) and a corresponding excess of the conversion homozygote in the cases compared with the control group (P=0.020).
In 486 subjects in whom urinary THaldo excretion rate was measured, both polymorphisms were noted to be in Hardy-Weinberg equilibrium. THaldo levels were higher in those subjects possessing the T allele of the SF-1 binding site and the conversion allele of the IC compared with those lacking these alleles (Table 4) (P=0.024). Subjects homozygous for T or heterozygotes (TC) had higher THaldo levels than those homozygous for C (P=0.05). However, the 3 genotypes for the IC were not significantly different.
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| Discussion |
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Our hypertensive group had a relative excess of the SF-1 site T allele and of the conversion allele of the IC. This is in agreement with the results of Soubrier (unpublished data, 1997). However, these findings contrast with those of Kupari et al16 who detected a significant association of the C allele and increased left ventricular mass. This was a cross-sectional study of normal subjects, and its relevance to raised BP is unclear.
Examination of the key intermediate phenotype, aldosterone levels, revealed that these alleles were also associated with higher excretion rates of the metabolite THaldo. This contrasts with the findings of Benetos et al27 who found higher supine plasma aldosterone concentrations in SF-1 site CC individuals than in TT individuals. Since the genes are in close linkage dysequilibrium, it is not possible to conclude which, if either, of the polymorphisms is responsible for differences in excretion between normotensive and hypertensive subjects.
If high aldosterone levels are associated with SF-1 (TT) and IC (CC) and if these alleles occur significantly more frequently in patients with essential hypertension (although no causal relationship can be assumed), it is perhaps permissible to speculate on mechanisms. For example, it is possible that these polymorphisms are associated with an altered relationship between Ang II and aldosterone and hypertension, but more detailed physiological studies will be necessary to ascertain this. These studies were not possible in the current investigation because we were unable to discontinue therapy or to control sodium intake in our ambulant outpatient population. It is also possible that the polymorphisms are in linkage with other causal mutations in neighboring genes. For example, the close proximity of CYP11B1 has already been mentioned, and there may be linkage to mutations in this gene that affect 11ßhydroxylase activity. To ascertain whether this relationship is indeed more sensitive in SF-1 (TT) and/or IC (CC) individuals, Ang II infusion studies or, at least, concurrent plasma renin and aldosterone levels will be necessary. It is relevant that Litchfield et al28 report relatively higher free cortisol excretion rates in essential hypertension; no mechanism to account for this has as yet been proposed.
In summary, we have observed an association between a polymorphism and the gene-encoding aldosterone synthase and hypertension in a careful case/control study. A possible intermediate phenotype has been identified on the basis of higher urinary aldosterone excretion in subjects bearing the allele that is overrepresented in patients with hypertension. Further studies of this locus to define the precise nature of the intermediate phenotype and the underlying physiological and clinical implications of this apparent association are now warranted.
| Acknowledgments |
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Received June 11, 1998; first decision July 14, 1998; accepted September 29, 1998.
| References |
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