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Division of Nephrology (S.G., J.N., B.D., F.J.F., B.M.F., P.F.) and Computational and Molecular Population Genetics Laboratory (G.L.), Zoological Institute, University of Berne, Berne 3010, Switzerland; Clinical Pharmacology Unit, Central University of Venezuela (I.S.H., A.M.C., L.X.C.), Caracas 1050, Venezuela; and Department of Nephrology, Fremantle Hospital, University of Western Australia (P.F.), Perth, Western Australia 6160, Australia
Address correspondence and requests for reprints to: Dr. Paolo Ferrari, School of Medicine and Pharmacology, University of Western Australia, and Department of Nephrology, Fremantle Hospital, Alma Street, Perth, Western Australia 6160, Australia. E-mail: paolo.ferrari{at}health wa.gov.au.
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Abstract |
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2 = 2.81; P = 0.09). In vivo 11ß-hydroxylase activity, estimated from urinary steroid profiling, was lower in subjects with an increased aldosterone to renin ratio or with the TconvGTA haplotype. These findings indicate that genotypes at the CYP11B locus are in strong LD and that identified haplotypes predict 11ß-hydroxylase activity. |
Introduction |
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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 (4, 5). Recent reports support the idea that variations at the gene (CYP11B2) that encodes this enzyme are associated with essential hypertension and may influence aldosterone secretion (6, 7, 8, 9, 10). Two variants, a cytosine/thymidine substitution in the 5' promoter region, which disrupts a putative binding site for steroidogenic factor-1 (SF-1), and a gene conversion in intron2 (Int2C) of CYP11B2, are in tight linkage disequilibrium (9, 10, 11). The combination of both variants in the SF-1 T/Int2C haplotype is increased in frequency in hypertensive patients (7) and has subsequently been associated with a high ARR in selected hypertensive patients (9, 10). The exact mechanisms for increased aldosterone synthase activity associated with the T allele in the CYP11B2 promoter are still the object of debate. The terminal steps in aldosterone synthesis in the zona glomerulosa of the adrenal are sequential 11ß-hydroxylation of deoxycorticosterone to yield corticosterone, followed by 18-hydroxylation and 18-dehydrogenation to produce aldosterone (12), which are all carried out by a single aldosterone synthase encoded by the CYP11B2 gene. Parallel 11ß-hydroxylation of the 17-hydroxysteroid, deoxycortisol, by the enzyme 11ß-hydroxylase produces cortisol. The gene CYP11B1 encodes this enzyme (12). Earlier data had shown that there is an unexplained increase in the secretion of 11-deoxysteroids in patients with hypertension (13, 14) and high plasma levels in patients with low renin hypertension (15). No molecular explanation for the apparent reduction in the efficiency of conversion of 11-deoxysteroid substrates to products in these subjects is available. However, the most recent findings of an association between the SF-1 polymorphism in CYP11B2 promoter and elevated deoxycortisol levels suggest that the variant in aldosterone synthase (CYP11B2) is linked to the reduced efficiency of adrenal 11ß-hydroxylation (16). In this study we investigated whether the 5' variant in CYP11B2 is in linkage with a key quantitative trait locus (QTL) in CYP11B1 that determines the above phenotype and whether subjects with high ARR would be the same individuals who have evidence of altered 11ß-hydroxylation efficiency. To validate the relevance of a potentially recognized QTL, a case study population from Venezuela was compared with an independent ethnic population from Switzerland.
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Subjects and Methods |
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For the initial screening of genetic markers at the CYP11B2/CYP11B1 locus, 40 subjects from the DNA database of the Division of Nephrology and Hypertension at University of Berne were selected on the basis of genotype for the polymorphism at the CYP11B2 SF-1 locus (10). Twenty were homozygous for the wild-type allele, and 20 for the mutant allele. These DNA samples were used to search for genetic variants in the promoter, intronic, and exonic regions of both CYP11B genes using an established method (17). In addition to the genetic variants identified by this method, other potential loci were searched in public databases (http://ncbi.nlm.nih.gov/SNP/ and http://snp.cshl.org/). This analysis yielded nine potentially informative polymorphisms identified at the CYP11B loci, of which eight were single nucleotide polymorphisms (SNPs), five in CYP11B2 and three in CYP11B1 (Fig. 1
). The previously described intron 2 conversion polymorphism in CYP11B2 (10) was also used as a genetic marker. Five SNPs were previously described by the National Center for Biotechnology Information, accessible on the dbSNP website (http://ncbi.nlm.nih.gov/SNP/). If available, the corresponding dbSNP identification number is reported.
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Population genetics data analysis was performed in two independent ethnic populations, one of which was also investigated for possible genotype-phenotype correlations. The case study population consisted of 99 healthy adult subjects from the Clinical Pharmacology Unit, University of Caracas (Caracas, Venezuela). Subjects with a known history of cardiac or cerebrovascular disease, evidence of renal or hepatic dysfunction, severe hypertension, impaired glucose tolerance or diabetes mellitus, age more than 70 yr, or taking birth control pills were excluded. A series of clinical, biochemical, and hormonal investigations, including blood pressure (BP) and heart rate measurements, serum electrolyte and creatinine, plasma renin and aldosterone studies, and 24-h urine collection, was performed. A urine aliquot was stored for urinary steroid analysis. BP was determined by sphygmomanometry after the subject had rested 30 min in the supine position. Korotkoff sounds 1 and 5 were used to record systolic and diastolic BP, respectively. The mean of three consecutive readings of systolic and diastolic BP was calculated and employed for statistical analysis. The control population consisted of 100 normal healthy subjects from University of Berne (Berne, Switzerland). These subjects were only used to statistically infer haplotypes at the CYP11B2-CYP11B1 locus from the identified genotypes as described below. Consent was obtained from the subjects in both populations for the respective studies.
Genotyping
All subjects from both populations were genotyped for the eight SNPs identified as well as the CYP11B2 intron 2 conversion polymorphism. Genotyping of the CYP11B2 intron 2 conversion and SF-1 polymorphisms was performed as previously described (10). The allelic discrimination assays, based on the fluorogenic 5'-nuclease activity, were adopted to genotype the other seven SNPs.
Primers and probes were designed and synthesized in conjunction with Applied Biosystems (Foster City, CA) by submitting the conserved target sequences. Each assay mix contained of forward and reverse primers and two minor groove binder probes; each probe was labeled with different fluorescent reporter dye (6-carboxy fluorescein or VIC) complementary to the wild-type and the variant allele. Taking into the consideration of the fact that both genes share approximately 95% identity in coding sequences, the specificity of the assays was obtained using the gene-specific templates amplified with specific primers (17). The assay target-specific primers and TaqMan MGB probes are reported in Table 1. Because of low allele frequency, CYP11B2 exon 5 A/G (rs4540) and CYP11B1 3'-untranslated region (3'UTR) A/G (rs5297) were discarded from the analysis in the case and control study populations; thus, only primers and probes eventually used for population analysis are reported (Table 1).
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Linkage disequilibrium (LD) and haplotypes computation
The pattern of LD at the CYP11B loci was measured using full genotype data obtained from 91 and 95 subjects in the case study and control population, respectively. The SNP frequencies were estimated using the maximum likelihood estimates (gene counting in the samples), and their SD was approximated with 10,000 bootstraps. The linkage between every pair of markers was tested in every sample using a simulated likelihood ratio test. Null distribution (no linkage) of the likelihood ratio was computed using the 10,000 permutations of alleles between individuals. Frequency estimations and LD tests were computed using the ARLEQUIN software (18, 19). The difference in the level of LD between the two samples was computed in a Bayesian setting using the Ayres and Balding method (20). For every pair of SNP, the probability that the LD in the South American is greater or lesser than that in the European group was deduced from the posterior distribution of D' in the two groups.
Currently available programs, such as PHASE (21), HAPLOTYPER (22), or ELB (23), were used to infer the multi-SNP phase of individuals and the haplotypes frequencies in the samples for the genes CYP11B2 and CYP11B1. Posterior distribution of the LD was computed between whole genes from these haplotypes frequencies and using the Ayres and Balding method (20) extended to a pair of loci with more than two alleles each.
Genotype/phenotype studies
The multi-SNP phase of individuals and the haplotype frequencies were also computed putting the gene CYP11B2 and CYP11B1 together using the software Phase (21). The distinct haplotype demonstrating LD between the polymorphism in the CYP11B2 defined above and variations in CYP11B1 was assessed for functional significance of the variation in 11ß-hydroxylase activity. The potential epistatic interaction between the two genes was investigated by examining the relationship between the two major haplotypes, markers of aldosterone excretion, and 11ß-hydroxylase activities in multifactorial regression models, which allowed simultaneous testing of relevant interaction effects and correction for appropriate covariates (age, body mass index, plasma potassium, etc.). This analysis was performed in the case population of healthy South American subjects.
Finally, we tested our subjects for the presence of a CYP11B2/CYP11B1 chimeric gene duplication (24), with a protocol established in our laboratory (17), to assess whether a given CYP11B haplotype was associated with the CYP11B2/CYP11B1 chimeric gene.
Analytical methods
Plasma renin activity was measured using the method described by Sealey et al. (25). The plasma aldosterone concentration was measured by the solid phase RIA technique using the PCP Coat-A-Count assay (Diagnostic Products Corp., Los Angeles, CA). The ARR was calculated, and an elevated ARR was defined using a cut-off of more than 500 pmol/liter·ng/ml·h, which is 70% of the published cut-off level for primary aldosteronism (26, 27).
Urinary markers of 11ß-hydroxylase activity were determined by gas chromatography/mass spectrometry analysis of the urinary steroid profile assessed by the method of Shackleton (28) modified in our laboratory (29). The 11ß-hydroxylase activity using urinary steroid analysis was calculated from the formula 100 x tetrahydro-11-deoxycortisol (THS)/[tetrahydroxycortisone (THE) + tetrahydro-11-deoxycortisol (THF) + 5
THF] as described by Shackleton (28), where THS is the excretory product of the cortisol precursor, deoxycortisol, and THE and THF are the tetrahydrometabolites of cortisone and cortisol, respectively.
Statistics
Gene frequencies and haplotype prevalences were calculated using ARLEQUIN software (18, 19), available at: http://lgb.unige.ch/arlequin/. Differences between means were assessed by t test or ANOVA for analysis of continuous variables and by nonparametric analysis for variables that were not normally distributed. Multifactorial regression models were used for simultaneous testing of relevant interaction effects and correction for appropriate covariates. Analyses were performed using the Systat 10.0 (SPSS, Inc., Chicago, IL) statistical software package. Values are expressed as the mean ± SD.
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Results |
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We identified a total of nine potentially informative loci; five SNPs in CYP11B2, three SNPs in CYP11B1, and the intron 2 conversion polymorphism of CYP11B2 (Fig. 1
). For CYP11B2, polymorphisms were the promoter SF-1 C/T (no. 1), intron 1 C/T (no. 2), intron 2 conversion (no. 3), exon 5 A/G (rs4540; no. 4), exon 6 T/G (rs4538; no. 5), and 3'UTR A/G (rs3097; no. 6). For CYP11B1, polymorphisms were the exon1 T/C (rs6410; no. 7), intron 6 G/A (no. 8), and 3'UTR A/G (rs5297; no. 9). Because of the low allele frequency (<0.1), SNPs 4 and 9 were discarded from the analysis in the case and control study populations. It was possible to obtain full genotype data from 91 subjects in the South American case study population and 95 subjects in the European control population. The allele frequencies of the retained (allele frequencies, >0.1) genetic markers are shown in Table 2. No marker exhibited a significant difference between Hardy-Weinberg equilibrium. SNPs 2–4 and 8 (intron 1, intron 2, and exon 6 of CYP11B2 and intron 6 of CYP11B1) showed allele frequencies significantly different (P < 0.05) between the two samples.
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All D' measured between pairs of SNP were highly significantly (P < 0.05, computed with 10,000 permutations and with Bonferroni correction for multiple tests) in both European and South American populations, showing that every pair of genetic markers is in LD (Table 3). More than one third of these pairwise comparisons (eight of 21) exhibited differences between the South American and European samples (Table 4). Interestingly, intron 1 of CYP11B2 exhibited different levels of LD between the two populations with almost all other markers. Five of eight significant pairwise comparisons were due to this SNP. The multi-SNP haplotype frequencies obtained with Phase (21) were consistent with those obtained with the method described by Excoffier (23).
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). These four haplotypes (A–D in Table 5) accounted for 76% of all 25 haplotypes from the seven markers tested in both populations. When two of the SNPs in the CYP11B2 (promoter SF-1 and 3'UTR) were omitted from the analysis, the variants intron 1C, intron 2 of the wild-type allele, and exon 6T of CYP11B2 were found to be in strong LD with exon 1C and intron 6G of CYP11B1 (P < 0.0001). Thus, this major haplotype (CwtTCG) was present in 38%, and the contrasting haplotype (TconvGTA) was present in 38% of Europeans, whereas in the South American population, the major haplotype was present in 48%, and the contrasting haplotype was present in 26% of subjects (Europeans vs. South Americans: 2 = 2.81; P = 0.09). Finally, we did not find any clear difference between samples when we used the reconstructed haplotypes to compute the LD between these two genes. The computed probability that the linkage between these two genes is higher in the European sample than in the South American sample was found to be approximately 0.7.
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In the 99 subjects of the South American case study population (average age, 40 ± 12 yr), systolic and diastolic BP averaged 116 ± 12 and 77 ± 10 mm Hg, respectively. The mean plasma renin activity was 3.9 ± 3.2 ng/ml·h, and plasma aldosterone was 393 ± 181 pmol/liter. An elevated ARR, defined as greater than 500 (pmol/liter·ng/ml·h) was found in 21 of the 99 subjects. When the urinary THS/(THE + THF + 5THF) ratio used to estimate in vivo 11ß-hydroxylase activity was analyzed according to the ARR, the ratio of these steroids was higher in subjects with high than with in those with normal ARR (P < 0.01), suggesting lower 11ß-hydroxylase activity associated with high ARR (Fig. 2). Substrate availability for 11ß-hydroxylase was comparable (THS, 24.4 ± 26.5 vs. 27.2 ± 24.9 µg/24 h, normal vs. high) in the two ARR groups. An increased ARR tended to be more prevalent with the TconvGTA than with the CwtTCG haplotype, but the difference did not reach statistical significance (2 = 3.39; P = 0.067). The urinary THS/(THE + THF + 5THF) ratio was further analyzed according to the identified haplotypes. This ratio was higher, albeit only slightly, in subjects with the TconvGTA than in those with the CwtTCG haplotype (P < 0.05), indicating lower 11ß-hydroxylase activity with the TconvGTA genotype (Fig. 2).
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Discussion |
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Current methods for testing LD are based on the assumption that haplotypes are observed, which can lead to erroneous associations. An estimate of the extent of LD that takes into account uncertainties in haplotype reconstruction was made using alternative Bayesian methods for inferring the extent of LD when haplotypes at different loci are inferred and not directly observed (21, 30). The method of Ayres and Balding (20) provides the posterior distribution of the LD in a given sample, which takes into account the uncertainty in the haplotype reconstruction between pairs of markers. This method deals with biallelic markers, such as SNPs, or with multiallelic markers, such as microsatellites or multi-SNP haplotypes. LD can be inflated by demographic factors, including inbreeding, population structure, and bottleneck, and also by a recent admixture of populations with different allele frequencies (31). We did not find a systematically higher LD in a given sample, which could have been ascribed to differences in the ethnic background of the two selected populations.
The findings of the present study demonstrate that genotypes at the CYP11B2-CYP11B1 locus are in strong LD. After estimating the multi-SNP haplotype frequencies in every gene, we directly used the method of Ayres and Balding extended to multiallelic loci to infer the global LD between the two genes (20). We showed that these two genes are in strong LD in every sample, which confirms what was found for every pairwise LD computation (20). Likewise, this method was not able to give any strong evidence of differences in LD between the two samples. However, in six of eight cases where a difference in LD was present, LD was stronger in the South American population, suggesting less variability in this population. Thus, the relevance of this genetic association is consolidated by the presence of characteristic CYP11B haplotypes demonstrated in two independent populations with different ethnic backgrounds.
Hereditary hypertension caused by chimeric gene duplications of CYP11B2/CYP11B1 and ectopic expression of aldosterone synthase was previously reported by Lifton et al. (24). We tested our population for the presence of the CYP11B2/CYP11B1 chimaeras using extra-long PCR (17). None was found to be carrier of this chimeric gene. Thus, none of the haplotypes identified is predictive of the presence of glucocorticoid remediable aldosteronism.
An increased ARR was recently related to a CYP11B2 polymorphism in the promoter region (7, 9, 10). However, the exact mechanisms for increased aldosterone synthase activity associated with the T allele in the CYP11B2 promoter are unclear. In vitro studies do not seem to support a pivotal role of altered aldosterone synthase activity. The T variant was found to influence binding of the transcriptional regulatory protein, SF-1, in vitro (32), with recent evidence suggesting that this change does not have any direct effect on transcriptional regulation (33). Detailed studies of the phenotype associated with the T allele of the SF-1 CYP11B2 promoter show that a striking and reproducible finding is the increased in production of 11-deoxysteroids, consistent with reduced 11ß-hydroxylase efficiency (16). To date, differences in circulating levels of 11-deoxycortisol have been used to infer altered 11-hydroxylase activity in subjects with increased ARR (16, 34, 35). Recently, Connell et al. (34) showed that the excretion of THS is heritable (19.4%) and that the T allele of CYP11B2 is more strongly associated with higher THS levels than the C allele of CYP11B2. We used the ratio of 11-deoxycortisol (substrate) to cortisol metabolites (product) to assess in vivo 11-hydroxyalse activity. Measuring urinary steroids and determining ratios do not equal actual determination of actual enzymatic activity, because substrate availability within the adrenal could be affected or clearance of steroids could be differentially affected. However, we consider this method to be more appropriate to assess in vivo 11-hydroxyalse activity (28) compared with circulating levels of a given steroid (for instance, 11-deoxycortisol), which is strongly dependent on production and clearance.
The reported increase in 11-deoxycortisol levels in association with the polymorphism in CYP11B2 could result from 1) end-product inhibition by aldosterone of CYP11B1, 2) competition for SF-1 transcription factor availability to CYP11B1 as well as other steroidogenic genes, and 3) LD with a QTL in CYP11B1. Our study confirms that decreased 11ß-hydroxylase activity is associated with the elevated ARR, a marker of aldosterone synthase activity. Moreover, it demonstrates that the two main CYP11B haplotypes identified are associated with differences in the urinary THS/(THE + THF + 5THF) ratio, suggesting that in vivo 11ß-hydroxylase activity is related to a QTL in CYP11B1. ACTH has been shown to affect aldosterone secretion, albeit as a temporary effect, in vivo (36). It has been proposed (34) that a primary effect of this would be a chronic, low grade increase in ACTH stimulation to the adrenal cortex. This may result in a resetting of the usual regulation of aldosterone, which, depending on interactions with other factors, would lead to hypertension associated with altered regulation of aldosterone.
In conclusion, our findings demonstrate that genotypes at the CYP11B2-CYP11B1 locus are in strong LD. These characteristic CYP11B haplotypes are demonstrated in two independent populations with different ethnic backgrounds. Identified haplotypes predict the urinary excretion ratio of THS/(THE + THF + 5THF), a marker for 11ß-hydroxylase activity.
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Footnotes |
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First Published Online October 26, 2004
Abbreviations: ARR, Aldosterone to renin ratio; BP, blood pressure; LD, linkage disequilibrium; QTL, quantitative trait locus; SF-1, steroidogenic factor-1; SNP, single nucleotide polymorphism; THE, tetrahydroxycortisone; THF, tetrahydro-11-deoxycortisol; THS, tetrahydro-11-deoxycortisol; UTR, untranslated region.
Received June 1, 2004.
Accepted October 15, 2004.
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