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Journal of Clinical Endocrinology & Metabolism , doi:10.1210/jc.2004-0870
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The Journal of Clinical Endocrinology & Metabolism Vol. 90, No. 2 1072-1077
Copyright © 2005 by The Endocrine Society

Genetic Variation at the Locus Encompassing 11-ß Hydroxylase and Aldosterone Synthase Accounts for Heritability in Cortisol Precursor (11-Deoxycortisol) Urinary Metabolite Excretion

Bernard Keavney, Bongani Mayosi, Nicole Gaukrodger, Helen Imrie, Michelle Baker, Robert Fraser, Mary Ingram, Hugh Watkins, Martin Farrall, Eleanor Davies and John Connell

Institute of Human Genetics (B.K., N.G., H.I., M.B.), University of Newcastle, Newcastle, United Kingdom; The Cardiac Clinic (B.M.), Department of Medicine, University of Cape Town, Cape Town, South Africa; Medical Research Council Blood Pressure Group (R.F., M.I., E.D., J.C.), Division of Cardiovascular and Medical Sciences, University of Glasgow, Glasgow, United Kingdom; and Department of Cardiovascular Medicine (H.W., M.F.), University of Oxford, Oxford, United Kingdom

Address all correspondence and requests for reprints to: Professor John Connell, MRC Blood Pressure Group, Western Infirmary, Glasgow G11 6NT, United Kingdom. E-mail: j.connell{at}clinmed.gla.ac.uk.


   Abstract
Top
 Abstract
Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
Genetic variation in the gene encoding aldosterone synthase (CYP11B2) has previously been shown to be associated with hypertension and left ventricular hypertrophy. The intermediate phenotype most consistently associated with variation at this locus is that of elevated plasma 11-deoxycortisol (S). However, in normal subjects, aldosterone synthase does not metabolize S, which is converted to cortisol (F) by the enzyme 11ß hydroxylase, encoded by the gene CYP11B1, which lies adjacent to CYP11B2 on chromosome 8. It is possible that the quantitative trait locus for the phenotype is within CYP11B1 and that linkage disequilibrium across the extended locus could account for these observations. However, variation across the whole CYP11B1/B2 locus had not been extensively characterized with respect to these phenotypes. We genotyped six polymorphisms in the CYP11B2 gene and three polymorphisms in the CYP11B1 gene in 248 Caucasian nuclear families comprising 1428 individuals. We measured plasma levels of S and F in 460 individuals from 86 families and urinary excretion rates of tetrahydrodeoxycortisol (THS) and tetrahydrodeoxycorticosterone in 573 individuals from 105 families. We examined heritability of the phenotypes and their association with genotypes and haplotypes at this locus.

All steroid phenotypes except urinary tetrahydrodeoxycorticosterone were highly heritable (P < 0.00001). There was strong linkage disequilibrium across the CYP11B1/B2 locus. There was modest evidence for association between polymorphisms of CYP11B2 and plasma levels of S (P = 0.02 for T4986C polymorphism) and the plasma S to F ratio, reflecting the activity of 11-ß hydroxylase (P = 0.01 for T4986C polymorphism). There was strong evidence for association between polymorphisms of both CYP11B1 and CYP11B2 and urinary THS, which was strongest for the CYP11B1 exon 1 polymorphism (P = 0.00002). Addition of other marker data to CYP11B1 exon 1 did not improve the fit of a log-linear model. Genotype at CYP11B1 explained approximately 5% of the variance in urinary THS excretion in the population. Thus, it is likely that linkage disequilibrium between causative CYP11B1 variants and CYP11B2 polymorphisms account for the previous observations. Further fine-mapping studies across the CYP11B1 locus are required to localize the causative variant(s) for the biochemical phenotype; this may also identify susceptibility alleles for hypertension and left ventricular hypertrophy.


   Introduction
Top
 Abstract
 Introduction
Subjects and Methods
 Results
 Discussion
 References
 
THE LATE STAGES in synthesis of aldosterone and cortisol (F) in the human adrenal cortex are catalyzed by the enzymes aldosterone synthase and 11-ß hydroxylase, respectively. The two enzymes share a high degree of amino acid homology and are encoded by genes that lie in tandem on chromosome 8 in man. CYP11B2 encodes aldosterone synthase and CYP11B1 11-ß hydroxylase. Variation across this locus accounts for the biochemical and cardiovascular phenotypes in the Dahl salt-sensitive hypertensive rat (1, 2). We have previously reported that polymorphic variation in the promoter region (–344C/T) of CYP11B2 is associated with essential hypertension and differences in left ventricular mass in man (3, 4, 5, 6, 7); some but not all studies in other populations have confirmed this finding (8, 9, 10). The mechanism that accounts for these final phenotypes is not defined. We have reported that this variation in CYP11B2 is particularly associated with a raised ratio of aldosterone to renin in hypertensive patients, suggesting that altered production of aldosterone is a key intermediate phenotype. However, in studies in nonhypertensive subjects, we and other groups have found that the most consistent association with the promoter polymorphism is an increase in plasma levels of S. This steroid has no biological activity but is the immediate precursor of F, being converted to the glucocorticoid by 11ß-hydroxylase. This association between variation in the gene encoding aldosterone synthase and the levels of the precursor of F appears initially paradoxical and requires more detailed evaluation. In particular, it is necessary to consider whether the observed association could arise from linkage disequilibrium between CYP11B2 polymorphisms and variants in the neighboring CYP11B1 gene (11, 12). Accordingly, we have examined the heritability of levels of plasma and urinary steroid metabolites and their association with polymorphic variation in the CYP11B1 and CYP11B2 genes in a large collection of nuclear families.


   Subjects and Methods
Top
 Abstract
 Introduction
 Subjects and Methods
Results
 Discussion
 References
 
Family collection

Caucasian families (248), comprising 1428 individuals, were recruited in the Oxford region of the United Kingdom between 1993–1997 as previously described (13, 14). Briefly, families were selected through a hypertensive proband with documented systolic and diastolic blood pressure in the top 5% of the population distribution either on multiple clinical readings or ambulatory blood pressure monitoring. To be suitable for the study, families were required to consist of at least three siblings (including the proband) clinically assessable for blood pressure if at least one parent of the sibship was available to give blood for DNA analysis and to consist of at least four assessable siblings (including the proband) if no parent was available for DNA analysis. Qualifying sibships could be either in the generation of the proband or his/her offspring, and there was no requirement for the sibship to contain additional members affected with hypertension (although this was not an exclusion criterion). Where members of the sibship were found to be hypertensive (using identical criteria to those applied in the probands), families were extended and the spouses and offspring of hypertensive sibs collected. Thus, the family collection includes some extended families, although most are nuclear families. In all cases, to minimize the chances of bilineal inheritance, only sibships where both parents were known not to be hypertensive were eligible for study. Blood pressure was measured using ambulatory monitoring for a period of 24 h (A&D TM2421, Takeda Medical, Tokyo, Japan) in all subjects willing to undergo monitoring. A full clinical history was taken, anthropometric measurements including height, weight, waist, and hip measurement were made, and blood was drawn (with the subject having been in a sitting position for 5 min before the blood draw) into a variety of anticoagulants for plasma and DNA analysis. Blood was not drawn at a particular time of day, although most families were visited in their homes in the evenings. Between 1998–2000, families were contacted again and asked to reattend for further phenotyping; at this visit, 12-lead electrocardiograms, echocardiograms, ultrasound measurement of carotid intima-medial thickness, and 24-h urine collections for steroid metabolites were performed. Urine was collected without preservative and aliquots stored at –20 C until analyzed.

Corticosteroid analysis

Plasma measurements of F and deoxycortisol (S) were made on the baseline bloods in 460 individuals from the first 86 families recruited into the study; these blood samples were taken at any time between 0900 and 1700 h. Plasma F concentration was measured by direct RIA (Coat-a-Count, Diagnostic Products Corporation Inc., Los Angeles, CA). Plasma S concentration was measured by RIA after partial purification by paper chromatography (15). The plasma S to F ratio was calculated as a conventional measure of 11-ß hydroxylase activity. The within- and between-batch coefficients of variation for these assays were 9.2 and 15.9% (S) and less than 5% (F). Twenty-four-hour excretion rates of tetrahydrodeoxycortisol (THS), the principal urinary metabolite of S, and tetrahydrodeoxycorticosterone (THDOC), the principal urinary metabolite of 11-deoxycorticosterone, were determined by gas chromatography-mass spectrometry using the method of Shackleton (16) with minor modifications. The lower limit of detection of the assay was 1.8 µg/liter. The inter- and intraassay coefficients of variation of the assay were 6.1 and 8.1% for THS and 11.2 and 12.9% for THDOC.

Genotyping

DNA was extracted using standard methods. Six biallelic polymorphisms that span the CYP11B2 gene were genotyped, as were three biallelic polymorphisms in exon 1, intron 3, and exon 8 of the CYP11B1 gene. The genomic location of each polymorphism is shown with respect to CYP11B1 and CYP11B2 exonic sequences in Fig. 1Go. The intron conversion polymorphism located in the second intron of the CYP11B2 gene that corresponds to a substitution of a portion of the second intron of CYP11B2 with that of CYP11B1, and a further five single nucleotide polymorphisms, T-344C, A2713G, A4550C, T4986C, and G5937A, were genotyped after PCR amplification and digestion by restriction enzymes as previously described (6).



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  		FIG. 1. Genomic location of each polymorphism.

 
Due to 95% homology between the CYP11B1 and CYP11B2 genes, a nested PCR methodology was adapted from Skinner et al. (17), using primer sequences described by White et al. (18), to genotype the CYP11B1 polymorphisms. To investigate the CTG to CTA polymorphism at codon 75 of exon 1, exons 1 and 2 were selectively amplified using the forward primer 5' TTT GAA TTC TCG AAG GCA AGG CAC CAG 3' and the reverse primer 5' GGG GGA TCC TGC TCC CAG CTC TCA GCT 3'. A 25-µL PCR containing 1.5 mM MgCl2 and 12.5 pmol each primer was denatured at 94C for 4 min, followed by 35 cycles of 94 C for 1 min, 65 C annealing for 1 min and 72 C extension for 3.5 min, and a final incubation of 72 C for 10 min. The resulting 1.3-kbp fragment was resolved on a 0.8% agarose gel. One microliter of this PCR product was then used in a nested PCR to specifically amplify exon 1 of CYP11B1. Using the same forward primer and the reverse primer 5' GAA TGG CAG TGC TGA GTG CC 3', the nested PCR conditions were as follows: a 93 C denaturation for 4 min, followed by 35 cycles of 93 C for 30 sec, annealing at 58 C for 30 sec, and extension at 72 C for 1 min, with a final extension at 72 C for 10 min. Five microliters of the resulting 388-bp PCR product was digested with the restriction enzyme AlwNI (5 U) at 37 C for a minimum of 2 h. Digested products were then resolved on 3.5% agarose gels with the G allele represented by bands at 154, 132, and 102 bp, and the A allele with bands at 256 and 132 bp.

To investigate the A to G transition 12 nucleotides 3' to the 5' end of intron 3, exons 3–5 were selectively amplified using the forward primer 5' CCC GAA TTC AGA AAA TCC CTC CCC CCT A 3' and the reverse primer 5' CCC GGA TCC GAC ACG TGG GCG CCG TGT GA 3'. PCR conditions were as described above to amplify a 1.7-kbp fragment. One microliter of this product was then used in a nested PCR to specifically amplify exons 3 and 4. The forward primer 5' CTG CAG GCC GAT TCC CCT TG 3' and the reverse primer 5' GTG GTG GAG AGG GAG AAA TT 3' amplified a 689-bp product in PCR conditions described above. Digestion with the restriction enzyme, NlaIII, resulted in bands of 312, 202, and 151 bp in the presence of the A allele and 514 and 151 bp in the presence of the G allele. To investigate the C to T polymorphism in exon 8, a 734-bp amplicon, encompassing exons 8 and 9 (partial), was amplified using the CYP11B1-specific forward primer, 5' TAC TCT CTG GGT CGC AAC CCC 3', and the specific reverse primer, 5' GGG GGC ACA TGC TGG GCC TCA 3'. A 25-µL PCR containing 1.5 mM MgCl2 and 12.5 pmol of each primer was denatured at 94 C for 3 min, followed by 35 cycles of 94 C for 1 min, 60 C for 1 min, and 72 C for 3 min and a final extension of 72 C for 10 min. Five microliters of the resulting product was digested with 1 U of the restriction enzyme BsoBI at 37 C overnight. Digested products were resolved on a 3% agarose gel with bands of 547 and 187 in the presence of the C allele. Genotyping was carried out blinded to the phenotypic information. Mendelian inheritance within families was confirmed using the PedCheck program (19), and inconsistencies were resolved by reexamination of the raw data and regenotyping where necessary.

Statistical methods

Exploratory analyses to test for normality of the phenotypic data, establish significant covariates, and remove outliers were performed using SPSS statistical software. Heritability of plasma F and S, and their ratio, and of urinary THS and THDOC was calculated using SOLAR (20). Identity-by-descent for each marker was assigned using MERLIN (21), and quantitative trait association analysis for individual polymorphisms was performed by a variance components approach using QTDT, with adjustment for significant covariates (22). Both total and within-family (orthogonal model) evidence for association was assessed. Marker allele frequencies, haplotype frequencies across the CYP11B1 and CYP11B2 genes, and pair-wise linkage disequilibrium were computed using FUGUE (23). To test for the presence of multiple quantitative trait loci at the locus, models incorporating two of the polymorphisms were constructed using PAP (24), and the log likelihoods of the two- and single-locus models were compared using a likelihood ratio test. A general review of analysis of multiple comparisons is available.


   Results
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
Discussion
 References
 
Twenty-four-hour urine collections were available in 573 individuals from 105 families, and plasma steroid measurements were available in 460 individuals from 86 families. The plasma levels of F and S and their ratio and the 24-h excretion of THS and THDOC are shown in Table 1Go. Because rigorous standardization for posture and time of blood sampling was not possible in this study, there would be an anticipated large influence of diurnal variation on the plasma measurements. Because the ratio between F and its precursor S may be less influenced by this and other factors, the S to F ratio was used as the principal plasma phenotype of interest. The plasma S to F ratio was highly skewed (because of the distribution of plasma S), so those individuals (53) with plasma S levels below the limit of detection were removed, and the ratio was then natural log-transformed before genetic analysis. Plasma levels of F and urinary THS and THDOC levels were only moderately positively skewed; therefore, analyses using both the untransformed and natural log transformed phenotypes were performed. Outliers >3SD from mean phenotypic values were excluded from analysis in all cases. Log-transformed plasma S and F levels were highly heritable phenotypes (45 and 37%, respectively, both P < 0.000001) as was the log-transformed S to F ratio (51%, P < 0.000001). Urinary excretion of THS was moderately heritable (19%; P < 0.000001), whereas heritability of THDOC did not differ significantly from zero. Females had significantly lower levels of THS than did males (P < 0.01); thus, sex was used as a covariate in the analysis of this phenotype. Age, body mass index, and blood pressure were not significant covariates of any phenotype. Because blood pressure was not significantly related to any of the phenotypes tested, no ascertainment correction was applied in the genetic analysis.


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  		TABLE 1. Characteristics of family members

 
The nine diallelic polymorphisms typed in CYP11B1 and B2 and their allele frequencies and heterozygosities within the population are shown in Table 2Go. All polymorphisms, with the exception of CYP11B1 exon 8 and CYP11B2 T4986C, had heterozygosities between 43–52%. There was no significant deviation from Hardy-Weinberg equilibrium for genotypes at any polymorphism in the pedigree founders. Table 3Go shows the extent of linkage disequilibrium (measured as D') between the polymorphisms, and Table 4Go shows the composition and frequencies of those haplotypes in the population with frequency >5%. Table 5Go shows the associations between CYP11B1 and CYP11B2 genotypes and plasma and urinary steroid phenotypes calculated using QTDT (orthogonal association model). There was weak evidence for association among the IC, A2713G, and T4986C polymorphisms and plasma S to F ratio, but no evidence for association between the CYP11B1 markers and plasma S to F ratio. With the exception of CYP11B1 exon 8 and CYP11B2 T4986C, all polymorphisms showed significant association with urinary THS excretion (in both log-transformed and untransformed analyses). The strength of the association was greatest with the CYP11B1 exon 1 polymorphism (P = 0.00002). When the effects of additional single markers were modeled together with CYP11B1 exon 1 in the two-marker PAP analyses, none of the two-marker models fitted the data significantly better than a model incorporating CYP11B1 exon 1 alone (Table 6Go). In the final CYP11B1 exon 1 model, the marker and the unlinked polygenes explained 5.3 and 30.4% of the total THS variability, respectively; each CYP11B1 exon 1 minor allele (A allele) was associated with a 35.7 nmol/24 h higher level of urinary THS excretion.


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  		TABLE 2. Characteristics of polymorphisms typed at the CYP11B1 and CYP11B2 loci

 

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  		TABLE 3. Linkage disequilibrium (measured as D') between nine polymorphisms spanning the CYP11B1 and CYP11B2 genes

 

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  		TABLE 4. Ranked haplotype frequencies (all haplotypes with frequency > 1%) in the CYP11B1 and B2 region

 

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  		TABLE 5. Association of various plasma and urinary steroid phenotypes with polymorphic markers at the CYP11B2 locus (QTDT analyses)

 

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  		TABLE 6. Two-marker analyses of association between genotypes and urinary THS levels incorporating CYP11B1 exon 1 and each of the other markers in turn

 

   Discussion
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
References
 
This study has confirmed the significant heritability of a number of plasma and urinary steroid phenotypes in a large collection of nuclear families and has shown for the first time that there is significant association between polymorphisms of the CYP11B1 gene and urinary excretion rates of the principal metabolite of S, THS. In previous work, we and others (3, 4, 25) had shown association between plasma levels of S and polymorphisms of the CYP11B2 gene. CYP11B2 is responsible for the final step in aldosterone synthesis, whereas the adjacent CYP11B1 gene converts S to F. A possible explanation for this association between a phenotype biologically more likely to arise from variability in the CYP11B1 gene and polymorphisms in the CYP11B2 gene was linkage disequilibrium between marker polymorphisms in CYP11B2 and causative variants in CYP11B1, which are separated by just 40 kbp on chromosome 8. In the present study, nine polymorphisms spanning the CYP11B1 and CYP11B2 locus, including the CYP11B2 polymorphisms previously shown to be associated with steroid phenotypes, were genotyped. We confirmed the existence of strong linkage disequilibrium across the region and correspondingly few common haplotypes. We have shown that evidence for association with urinary THS levels is strongest with the exon 1 polymorphism of CYP11B1. When genotype at this polymorphism was included in two-marker models, there was no evidence for additional contributions from any of the other markers in CYP11B1 or CYP11B2 that were typed. Thus, our results strongly suggest that the association observed in this and other studies between differences in 11ß-hydroxylation and polymorphisms in this region arises from a causative variant or variants in the CYP11B1 gene.

Our heritability analysis shows that plasma levels of S and F and their ratio (a frequently used clinical index of 11-ß hydroxylase activity) and urinary excretion of the principal metabolite of S, THS, are all heritable phenotypes. These findings are consistent with previous data reported in an adult twin population (26). In contrast, urinary THDOC excretion rate was not significantly heritable. This is the urinary metabolite of 11-deoxycorticosterone, which is produced mainly in zona fasciculata and is the immediate precursor to corticosterone. Conversion of S to F and of 11-deoxycorticosterone to corticosterone in zona fasciculata are both regulated by the enzyme 11ß-hydroxylase, so similar heritability and association of urinary THDOC with CYP11B1 polymorphisms might have been expected. Failure to demonstrate heritability of THDOC may partly be accounted for by lack of measurement sensitivity; excretion rate was below the limit of detection in a significant percentage of subjects.

We showed moderate evidence for association between polymorphisms in CYP11B2, although not CYP11B1, and log-transformed plasma S levels. Perhaps surprisingly, there was only weak association among the A4550C and T4986C polymorphisms of CYP11B2 (and none between the CYP11B1 polymorphisms) and the log-transformed plasma S to F ratio, which in a similar fashion to urinary THS levels indicates the activity of CYP11B1. Blood sampling times in this study were not standardized; the narrow range of variation and the inherent pulsatility of plasma concentrations may be responsible. Our results with urinary THS levels suggest that, where such standardized sampling is not possible, 24-h urinary collections provide a robust alternative index of steroid 11ß-hydroxylation. Moreover, in normal variation of efficiency, as opposed to clinical deficiency, final product levels (i.e. F and corticosterone) will be determined by variation in normal metabolic demand, whereas precursor levels (i.e. S and 11-deoxycorticosterone) will reflect the ACTH drive necessary to achieve final product levels. Therefore, precursor levels rather than precursor to product ratios may better reflect small, normal variations in 11ß-hydroxylase efficiency.

The exon 1 polymorphism we have typed in CYP11B1 is a change from CTG to CTA at codon 75; this change has no consequences for the amino acid sequence of the final protein because leucine is encoded in both cases. Thus, there is no obvious mechanism by which this polymorphism can influence the trait. Further studies will be needed to identify the causative variant. Fewer polymorphisms in CYP11B1 than in CYP11B2 have been described to date, and the present study suggests that a comprehensive cataloguing of genetic variation across this locus is now indicated to attempt to map the causative genetic variant precisely. Because linkage disequilibrium across the locus is strong in Caucasians, it is likely that trans-ethnic approaches incorporating populations of African origin, in whom the extent of linkage disequilibrium is likely to be less, will be useful in the eventual localization of the variant(s), as we previously showed in work at the angiotensin-1 converting enzyme locus (27).

Polymorphisms in CYP11B2 have been associated with essential hypertension in some but not all studies. We have previously reported that this is most marked in subjects with a raised aldosterone to renin ratio, in whom there is evidence of mineralocorticoid excess (4). The biological significance of phenotypes indicating reduced 11ß-hydroxylase activity, such as an increased plasma S to F ratio, or an elevated urinary THS level, remains uncertain. Previous studies in patients with hypertension have reported that plasma levels of S are raised. At present there are no large-scale data on urinary THS levels in patients with hypertension and in particular in those subjects with a raised aldosterone to renin ratio. In contrast with CYP11B2, which has been investigated thoroughly in essential hypertension, no large study has examined the association between CYP11B1 variants and blood pressure. The present study, which confirms that the intermediate phenotype is likely to arise because of genetic variation in CYP11B1, suggests that such studies would be timely.

The raised levels of S and of THS excretion are consistent with relative inefficiency of 11ß-hydroxylation within the adrenal zona fasciculata. Whether this mechanism directly contributes to the development of hypertension is unclear, but it is consistent with findings in hypertensive patients (28). We have speculated elsewhere that a genetically determined mild decrease in 11ß-hydroxylase activity may, through negative feedback, result in chronic (lifelong) minor activation of the hypothalamic/pituitary/adrenal axis. The net effect of this would be to expose the adrenal cortex to increased ACTH and other proopiomelanocortin-derived peptides over many years. This may be relevant to the pathogenesis of essential hypertension and, in particular, to the development of relative aldosterone excess (29). Further studies to define fully the intermediate phenotype in patients with hypertension and to examine the relationship between this phenotype and variation across the CYP11B2/CYP11B1 locus are currently in progress.


   Acknowledgments
 
We thank Anna Zawadzka, Polly Whitworth, and Jane Cadd for assistance with family collection.


   Footnotes
 
First Published Online November 2, 2004

Abbreviations: F, Cortisol; S, 11-deoxycortisol; THS, tetrahydrodeoxycortisol.

Received May 14, 2004.

Accepted October 26, 2004.


   References
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 

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