Promoter variants of aldosterone synthase gene (CYP11B2) and salt-sensitivity of blood pressure

The T(-344)C polymorphism in promoter of CYP11B2 gene encoding aldosterone synthase has been associated with differences in plasma aldosterone (ALDO) concentrations. In addition, the results of recent study carried out in Japan suggest that C(-344) allele of CYP11B2 may be a genetic marker of salt-sensitive hypertension characterized by low plasma renin activity (PRA) and high ALDO/PRA ratio. Therefore, it raises the question of whether the T(-344)C polymorphism of CYP11B2 gene may be associated with salt-sensitive hypertension in Caucasians. The DNA samples were obtained from 68 Polish hypertensives. During 3 subsequent 1-week periods each subject received diets of normal, low and high sodium content (120-140, 20-40 and 240-260 mmol Na+/day, respectively). Salt sensitivity was expressed as the difference between mean arterial pressure (MAP) on high salt diet and MAP on low salt one (delta MAPH-L). Genomic DNA isolated from peripheral blood nuclear cells was amplified by PCR method with primers flanking the polymorphic region and C(-344) allele was identified by gain of Hae III restriction site. There were 14 TT homozygotes (20.6%), 35 TC heterozygotes (51.5%) and 19 CC homozygotes (27.9%) in the studied group. No significant differences in delta MAPH-L, glomerular filtration rate, natriuresis, excreted fraction of filtered sodium, PRA, ALDO and ALDO/PRA ratio determined on each diet have been found in subjects according to CYP11B2 genotype. Our preliminary results suggest the lack of association of the T(-344)C CYP11B2 polymorphism with salt-sensitive hypertension as well as with activity of plasma renin-angiotensin-aldosterone system in Caucasian patients.     

Haplotype analysis of aldosterone synthase gene (CYP11B2) polymorphisms shows association with essential hypertension

OBJECTIVE: The CYP11B2 locus is an important candidate region in essential hypertension (HT). We therefore investigated CYP11B2 polymorphisms T-344C, T4986C and A6547G for association with essential HT. This included haplotype analysis and measurement of plasma aldosterone levels. METHODS: The three single nucleotide polymorphisms were genotyped by polymerase chain reaction-restriction fragment length polymorphism analysis of genomic DNA from 146 HT and 291 normotensive (NT) white subjects of Anglo-Celtic descent, in whom parental blood pressure status was the same as the subjects'. Genotype and allele frequencies in HTs and NTs were compared by chi2 analysis. Linkage disequilibrium and haplotype frequencies were estimated by the program 'snphap'. Phenotype-genotype relationships were tested using one-way analysis of variance. RESULTS: The T-344C variant was associated with HT (chi2 = 7.4, P = 0.0064). This association was confined to female HTs (P = 0.0061 for genotypes, P = 0.0013 for alleles). A strong association with HT was also seen for the A6547G variant (P = 0.0015), being greatest in females (P < 0.0001). No association was seen for the T4986C variant. Haplotype analysis of the three single nucleotide polymorphisms across eight different haplotype combinations showed a significant association with HT (chi2 = 24, seven degrees of freedom, P < 0.001). No significant tracking of plasma aldosterone with genotype was observed. CONCLUSION: The T-344C and A6547G, but not the T4986C, variants of the aldosterone synthase gene are associated with HT in females of the Anglo-Celtic population studied. This was reinforced by haplotype analysis.     

Effects of genetic variation in the aldosterone synthase (CYP11B2) gene on enzyme function

Objective  Evidence suggests that high levels of aldosterone lead to hypertension and increased risk of cardiovascular disease. Around 15% of patients with essential hypertension have a raised aldosterone to renin ratio (ARR) suggesting that aldosterone production is inappropriately high in relation to its principal agonist angiotensin II. This may be due to increased activity of aldosterone synthase caused by genetic variation in the CYP11B2 gene. We screened the coding region of human CYP11B2 for genetic variants and tested their effects on function in vitro .
Protocol  Normotensive subjects ( n  = 69) were screened for sequence variants in the coding region of CYP11B2 by single-stranded conformation polymorphism (SSCP) analysis and sequencing. The effects of nonsynonymous variants on enzyme activity were assessed in JEG-3 cells transiently transfected with wild-type or variant expression plasmids. The conversion of the substrate 11-deoxycorticosterone (DOC) to corticosterone (B) and aldosterone was measured.
Results  Twenty variants were detected in CYP11B2 and eight analysed functionally (Arg87Gly, Asn281Thr, Gly288Ser, Lys296Asn, Asp335Asn, Gln404Arg, Ala414Pro and His439Tyr). Corticosterone synthesis was unaltered and aldosterone synthesis reduced in variant Arg87Gly; Asn281Thr increased corticosterone and decreased aldosterone production; Gly288Ser increased corticosterone production and abolished aldosterone production; Lys296Asn reduced both corticosterone and aldosterone production; Asp335Asn increased corticosterone synthesis but did not affect aldosterone production. Variants Gln404Arg, Ala414Pro and His439Tyr showed increases in both corticosterone and aldosterone synthesis compared to the wild-type.
Conclusion  The study confirms the genetic variability of the CYP11B2 gene and provides us with additional valuable structure–function information.     

Congenital hyperreninemic hypoaldosteronism unlinked to the aldosterone synthase (CYP11B2) gene.

Isolated hyperreninemic hypoaldosteronism presenting in infancy is usually caused by mutations in the CYP11B2 gene encoding aldosterone synthase. We studied five patients in four unrelated kindreds with hyperreninemic hypoaldosteronism, in whom we were unable to find such mutations. All presented in infancy with failure to thrive, hyponatremia, hyperkalemia, markedly elevated plasma renin activity, and low or inappropriately normal aldosterone levels. All had normal cortisol levels and no signs or symptoms of congenital adrenal hyperplasia. All responded to fludrocortisone treatment. There were no mutations detected in exons or splice junctions of CYP11B2. Linkage of the disorder to CYP11B2 was studied in two unrelated consanguineous patients and in an affected sib pair. The consanguineous patients were each heterozygous for at least one of three polymorphic microsatellite markers near CYP11B2, excluding linkage to CYP11B2. However, linkage of the disease to CYP11B2 could not be excluded in the affected sib pair. Genes involved in the regulation of aldosterone biosynthesis, including those encoding angiotensinogen, angiotensin-converting enzyme, and the AT1 angiotensin II receptor were similarly excluded from linkage. These results demonstrate the existence of an inherited form of hyperreninemic hypoaldosteronism distinct from aldosterone synthase deficiency. The affected gene(s) remain to be determined.     

Evaluation of the aldosterone synthase (CYP11B2) gene polymorphism in patients with myocardial infarction

Left ventricular remodeling after myocardial infarction involves activation of the renin-angiotensin-aldosterone system. Recently, the biallelic -344T/C polymorphism of the aldosterone synthase gene was associated with increased aldosterone levels, arterial hypertension, diastolic dysfunction, and left ventricular dilatation. We hypothesized that this polymorphism may also affect left ventricular geometry and function after myocardial infarction. By using a standardized questionnaire, as well as anthropometric and echocardiographic measurements, we thus studied 606 patients (533 men and 73 women) who had a myocardial infarction before the age of 60 years. The aldosterone synthase gene polymorphism was analyzed after polymerase chain reaction amplification and restriction enzyme digestion. The results demonstrated that there was no association of the aldosterone synthase gene polymorphism with echocardiographically determined left ventricular dimensions, wall thicknesses, or indexes of systolic or diastolic function. Furthermore, anthropometric data, including blood pressure levels, were balanced between the different genotypes. Finally, the allele frequency was similar for patients with myocardial infarction and a sample group from the normal population (n=1675). The data indicate that the allele status of the aldosterone synthase gene polymorphism is not useful for the identification of patients with myocardial infarction who have impaired left ventricular function or unfavorable remodeling.     

Aldosterone synthase gene (CYP11B2) C-344T polymorphism, plasma aldosterone, renin activity and blood pressure in a multi-ethnic population

BACKGROUND: The aldosterone synthase gene (CYP1B2) locus is a candidate region involved in the development of hypertension. OBJECTIVE: To study the relationship between the C-344T CYP1B2 polymorphism, plasma aldosterone, renin activity and blood pressure in a multi-ethnic population. DESIGN: Population-based, cross-sectional study of 1313 middle-aged men and women (456 white, 441 of African origin and 416 South Asian). Anthropometry, blood pressure, biochemistry, questionnaire data and timed urine collections were taken with standardized techniques. All were genotyped for the C-344T CYP11B2 polymorphism. RESULTS: The frequency of the C allele was significantly lower in people of African origin (0.21) than in white (0.46) and South Asian (0.43) (P < 0.001). After adjustment for age, sex and ethnicity the TT genotype was associated with 14% higher plasma aldosterone levels, 3.7 mmHg higher systolic and 2.1 mmHg higher diastolic blood pressure than CC (P for linear trend < 0.05). No significant interactions with age, sex, ethnicity, body mass index (BMI) and fractional excretion of sodium were found in the associations between genotype and both blood pressure and aldosterone levels. In a sub-sample of participants in which plasma renin activity was measured (n = 457), a significant excess of T alleles was found in those with a raised (>/= 750) aldosterone-to-renin ratio (ARR). CONCLUSION: In this multi-ethnic population, the C-344T CYP1B2 polymorphism is associated with blood pressure, plasma aldosterone levels and ARR. Although significant differences in allele frequencies were found between groups, ethnicity does not explain the results.     

Aldosterone excretion rate and blood pressure in essential hypertension are related to polymorphic differences in the aldosterone synthase gene CYP11B2

Significant correlation of body sodium and potassium with blood pressure (BP) may suggest a role for aldosterone in essential hypertension. In patients with this disease, the ratio of plasma renin to plasma aldosterone may be lower than in control subjects and plasma aldosterone levels may be more sensitive to angiotensin II (Ang II) infusion. Because essential hypertension is partly genetic, it is possible that altered control of aldosterone synthase gene expression or translation may be responsible. We compared the frequency of 2 linked polymorphisms, one in the steroidogenic factor-1 (SF-1) binding site and the other an intronic conversion (IC), in groups of hypertensive and normotensive subjects. In a larger population, the relationship of aldosterone excretion rate to these polymorphisms was also evaluated. In 138 hypertensive subjects, there was a highly significant excess of TT homozygosity (SF-1) over CC homozygosity compared with a group of individually matched normotensive control subjects. The T allele was significantly more frequent than the C allele in the hypertensive group compared with the control group. Similarly, there was a highly significant relative excess of the conversion allele over the "wild-type" allele and of conversion homozygosity over wild-type homozygosity in the hypertensive group compared with the control group. In 486 subjects sampled from the North Glasgow Monitoring of Trends and Determinants in Cardiovascular Disease (MONICA) population, SF-1 and IC genotypes were compared with tetrahydroaldosterone excretion rate. Subjects with the SF-1 genotypes TT or TC had significantly higher excretion rates than those with the CC genotype. The T allele was associated with higher excretion rates than the C allele. However, no significant differences were found in excretion rate between subjects of different IC genotype. Urinary aldosterone excretion rate may be a useful intermediate phenotype linking these genotypes to raised BP. However, no causal relationship has yet been established, and it is possible that the polymorphisms may be in linkage with other causative mutations.     

Mutations in the human CYP11B2 (aldosterone synthase) gene causing corticosterone methyloxidase II deficiency.

Corticosterone methyloxidase II (CMO-II) deficiency is an autosomal recessive disorder of aldosterone biosynthesis, characterized by an elevated ratio of 18-hydroxycorticosterone to aldosterone in serum. It is genetically linked to the CYP11B1 and CYP11B2 genes that, respectively, encode two cytochrome P450 isozymes, P450XIB1 and P450XIB2. Whereas P450XIB1 only catalyzes hydroxylation at position 11 beta of 11-deoxycorticosterone and 11-deoxycortisol, P450XIB2 catalyzes the synthesis of aldosterone from deoxycorticosterone, a process that successively requires hydroxylation at positions 11 beta and 18 and oxidation at position 18. To determine the molecular genetic basis of CMO-II deficiency, seven kindreds of Iranian-Jewish origin were studied in which members suffered from CMO-II deficiency. No mutations were found in the CYP11B1 genes, but two candidate mutations, R181W and V386A, were found in the CYP11B2 genes. When these mutations were individually introduced into CYP11B2 cDNA and expressed in cultured cells, R181W reduced 18-hydroxylase and abolished 18-oxidase activities but left 11 beta-hydroxylase activity intact, whereas V386A caused a small but consistent reduction in the production of 18-hydroxycorticosterone. All individuals affected with CMO-II deficiency were homozygous for both mutations, whereas eight asymptomatic subjects were homozygous for R181W alone and three were homozygous for V386A alone. These findings confirm that P450XIB2 is the major enzyme mediating oxidation at position 18 in the adrenal and suggest that a small amount of residual activity undetectable in in vitro assays is sufficient to synthesize normal amounts of aldosterone.     

Haplotype association and synergistic effect of human aldosterone synthase (CYP11B2) gene polymorphisms causing susceptibility to essential hypertension in Indian patients

Background: Aldosterone synthase (CYP11B2) is a key enzyme involved in the terminal steps of aldosterone biosynthesis. Genetic variability in CYP11B2 gene has been associated with heterogeneous aldosterone production, which can affect sodium homeostasis and thereby regulation of blood pressure. Hence, the present study was aimed to explore the single-locus variations, haplotype and epistasis patterns of CYP11B2 (C-344T, intron-2 gene conversion and Lys173Arg) gene polymorphisms, and the risk contributed by them to the development of essential hypertension (EHT). Methods: A total of 279 hypertensive patients and 200 normotensive controls were enrolled in this study. C-344T and Lys173Arg polymorphisms of CYP11B2 gene were genotyped by PCR-RFLP method and intron-2 gene conversion (IC) polymorphism by allele-specific PCR analysis. Results: Single-locus analysis revealed significant association of CYP11B2 C-344T and Lys173Arg polymorphisms with EHT (p < 0.05). Considering the sexes, Lys173 allele was found to be at risk for hypertension in males (OR 1.40; 95% CI = 1.01–1.96). Unphased haplotype analysis revealed H1 (T-Conv-Lys; p = 0.0017) to have significant risk for EHT, while haplotype H4 (T-Wt-Arg) had a significant protective effect. Multifactor dimensionality reduction (MDR) interaction analysis found the overall best model with C-344T and IC polymorphisms exhibiting strong synergistic effect. Conclusion: The present study revealed a strong synergistic effect of CYP11B2 C-344T and IC polymorphisms causing susceptibility to EHT and haplotype H1 (-344T-Conv-Lys173) as the risk-conferring factor for hypertension predisposition.     

Deletion hybrid genes, due to unequal crossing over between CYP11B1 (11beta-hydroxylase) and CYP11B2(aldosterone synthase) cause steroid 11beta-hydroxylase deficiency and congenital adrenal hyperplasia.

Chromosomal rearrangements are natural experiments that can provide unique insights into in vivo regulation of genes and physiological systems. We have studied a patient with congenital adrenal hyperplasia and steroid 11beta-hydroxylase deficiency who was homozygous for a deletion of the CYP11B1 and CYP11B2 genes normally required for cortisol and aldosterone synthesis, respectively. The genes were deleted by unequal recombination between the tandemly arranged CYP11B genes during a previous meiosis, leaving a single hybrid gene consisting of the promoter and exons 1-6 of CYP11B2 and exons 7-9 of CYP11B1. The hybrid gene also carried an I339T mutation formed by intracodon recombination at the chromosomal breakpoint. The mutant complementary DNA corresponding to this gene was expressed in COS-1 cells and was found to have relatively unimpaired 11beta-hydroxylase and aldosterone synthase activities. Apparently the 11beta-hydroxylase deficiency and the adrenal hyperplasia are due to the lack of expression of this gene in the adrenal zona fasciculata/reticularis resulting from replacement of the CYP11B1 promoter and regulatory sequences by those of CYP11B2.     

Haplotypes of aldosterone synthase (CYP11B2) gene in the general population of Japan: the Ohasama study.

Since the identification of a chimeric aldosterone synthase which induces mendelian hypertension, polymorphisms in aldosterone synthase (CYP11B2) has been one of major targets for molecular analyses in association with hypertension. To date, four polymorphic variants of CYP11B2, -344T/C at promoter region, a gene conversion in intron 2, 2713A/G (in exon 3) which converts from Lys to Arg at codon 173 (K173R), and 4986T/C (in exon7) which converts from Val to Ala at codon 386 (V386A), have been identified in Caucasian population. Then, linkage disequilibrium between -344T/C polymorphism and a gene conversion in intron 2 or K173R mutation has been described, suggesting the presence of genetic haplotypes in Caucasians. Since the presence of a gene conversion in intron 2 or V386A mutation was still unknown in the Japanese population, all these polymorphisms were examined together to determine the CYP11B2 haplotypes of Japanese, using DNA samples from 1290 participants of the Ohasama study, who represent the general population of a rural community of northern Japan. Molecular analyses demon- strated the presence of a gene conversion of intron 2, but the absence of V386A mutation in Japanese population. The complete linkage disequilibrium between -344T/C polymorphism and K173R mutation was noted. Although -344T allele was linked either with a gene conversion in intron 2 or with normal intron 2, -344C allele was completely linked with normal intron 2. These results indicate the presence of 3 allelic haplotypes of CYP11B2, -344C with normal intron 2 and 173R, -344T with normal intron 2 and 173K, and -344T with converted intron 2 and 173K, in the general Japanese population. The frequency (total 1.0) was 0.35, 0.53, and 0.12, respectively. The presence of allelic haplotypes is considered to be an additional genetic information to individual polymorphism of CYP11B2 to determine the linkage between CYP11B2 polymorphisms and hypertension.     

CYP11B2 gene polymorphisms in idiopathic hyperaldosteronism

Primary aldosteronism is characterized by autonomous production of aldosterone and arterial hypertension, and it occurs in 2 principal forms: aldosterone-producing adenoma (APA) and idiopathic hyperaldosteronism (IHA). APA can be cured through removal of the adenoma, whereas IHA leads to hypertension that must be treated with medication. The origin of the autonomous aldosterone production in IHA is poorly understood, but genetic factors may contribute to its cause. To test the hypothesis that variants of the aldosterone synthase gene may contribute to susceptibility to IHA, we compared genotypes at 3 polymorphic sites in the CYP11B2 gene in patients with IHA (n=90) with those found in patients with APA (n=38), in patients with essential hypertension (n=72), and in normotensive individuals (n=102). We observed significant linkage disequilibrium among the 3 polymorphisms with 2 frequent haplotypes in all groups studied. One haplotype (C2R) was found to be increased in frequency in the IHA group (47%) compared with the other groups, which had a similar haplotype frequency (36%). The 3 polymorphisms studied have been implicated in either essential hypertension or excess aldosterone production in previous studies. Because of the strong linkage disequilibrium, the observed results could be due to the action of any 1 of the 3 alleles or to another allele in linkage disequilibrium with them. Our results suggest that variations in the CYP11B2 gene may contribute to dysregulation of aldosterone synthesis and lead to susceptibility to IHA.     

Association between aldosterone production and variation in the 11beta-hydroxylase (CYP11B1) gene

CONTEXT: Variation in the region of chromosome 8 including the genes steroid 11beta-hydroxylase (CYP11B1) and aldosterone synthase (CYP11B2) influences mineralocorticoid and glucocorticoid metabolism. However, the relative importance of polymorphisms in CYP11B1 and CYP11B2 in determining these phenotypes is unknown. OBJECTIVE: Our objective was to investigate genetic influences of the CYP11B1 and CYP11B2 genes on mineralocorticoid metabolism. DESIGN: We measured 24-h urinary excretion of the key metabolites of the principal mineralocorticoids, glucocorticoids and androgens secreted by the adrenal cortex. We genotyped polymorphisms spanning the CYP11B1 and CYP11B2 genes, which together capture all common variations at the locus. PARTICIPANTS: Participants included 573 members of 105 British Caucasian families ascertained on a hypertensive proband. MAIN OUTCOME MEASURES: We assessed heritability of urinary tetrahydroaldosterone (THAldo) excretion and association of THAldo excretion with genotype. RESULTS: The heritability of THAldo excretion was 52% (P < 10(-6)). There was significant association between THAldo and genotype at several of the CYP11B1/B2 polymorphisms. The strongest association was observed at the rs6387 (2803A/G) polymorphism in intron 3 of CYP11B1 (P = 0.0004). Association followed a codominant model with a 21% higher THAldo excretion per G allele. Genotype at rs6387 accounted for 2.1% of the total population variability of THAldo. We found significant association between THAldo excretion and urinary total androgen excretion, urinary tetrahydrodeoxycortisol level, and urinary cortisol metabolites (all P < 0.001). CONCLUSIONS: Aldosterone synthesis is highly heritable and is affected by genotype at CYP11B1. Our findings support the hypothesis that genetically determined differences in 11-hydroxylation efficiency can have downstream effects on mineralocorticoid synthesis. Such effects may be of relevance to the development of low-renin essential hypertension.     

Analysis of promoter region polymorphism in the aldosterone synthase gene (CYP11B2) as a risk factor for myocardial infarction

Several polymorphisms in genes of the reninangiotensin-aldosterone system have been found to have pleiotropic effects on cardiovascular disorders. Recently, a polymorphism (-344 C/T) in the promoter region of the aldosterone synthase gene (CYP11B2), which may influence plasma aldosterone levels, has been reported to strongly influence left ventricular diameters and mass in young adults and arterial stiffness in essential hypertensives. We investigated any association with risk of myocardial infarction (MI). CYP11B2 -344 polymorphism genotypes were determined by polymerase chain reaction (PCR) in 542 acute MI cases and 500 control subjects without history of coronary disease. All subjects were white and <75 years old. There was no significant difference in either genotype distributions (cases CC 17%, CT 52%, TT 31%; controls CC 22%, CT 47%, TT 31%, P = .10) or allele frequencies (cases C/T 0.43/0.57, controls C/T 0.46/0.54, P = .39) between cases and controls. The odds ratio (OR) for MI associated with the CC genotype was 0.75 (0.54-1.05), and remained insignificant when analysis was restricted to the 129 (24%) cases and 193 (37%) controls < 55 years of age (OR 0.68 [0.36-1.27], P = .20). In further analyses, there was no interaction of the polymorphism with other cardiovascular risk factors (smoking, hypertension, diabetes, body mass index, or cholesterol level) in determining MI risk, and the polymorphism did not influence the frequency of these risk factors in either cases or controls. In the case cohort, age at MI was not significantly different in subjects with the three genotypes (CC 61.2 +/- 9.8 years, CT 61.8 +/- 9.1 years, TT 62.2 +/- 9.0 years, P = .69). We conclude that the aldosterone synthase -344 promoter region polymorphism does not significantly influence the risk of MI either directly or via interaction with other risk factors.     

Association between aldosterone synthase (CYP11B2) polymorphism and left ventricular mass in human essential hypertension

OBJECTIVES: The aim of our study was to evaluate the relationship between aldosterone synthase gene polymorphism and cardiac dimensions in essential hypertension. BACKGROUND: Higher aldosterone synthase messenger ribonucleic acid levels in the human heart are accompanied by increased intracardiac aldosterone production, a phenomenon that is associated with cardiac fibrosis and hypertrophy. Recent evidence suggests that a polymorphism (-344C/T) in the promoter region of the aldosterone synthase gene is associated with increased constitutive aldosterone production. METHOD: Relationships between M-mode echocardiographic cardiac dimensions and aldosterone synthase -344C/T polymorphism were studied in 210 never-treated, middle-aged patients (age 41.6 +/- 1.4 years) affected by mild to moderate essential hypertension. Among these patients, 48 had the genotype -C344C, 97 had -C344T, and 65 had -T344T. Patients in the three groups were similar in terms of age, gender, body mass index, and blood pressure. RESULTS: Left ventricular (LV) mass and thickness were positively correlated with the number of T alleles: LV mass (CC, CT, and TT, respectively: 168 +/- 6.9, 179 +/- 5.2, and 193 +/- 6.9 g; p = 0.03), LV septal thickness (0.99 +/- 0.02, 1.03 +/- 0.02, and 11.08 +/- 0.03 cm, p = 0.04), PWT (0.93 +/- 0.03, 0.95 +/- 0.01, and 1.03 +/- 0.02 cm; p = 0.002), and relative wall thickness (38.3 +/- 1.2%, 38.8 +/- 0.8%, and 42.8 +/- 1.1%; p = 0.004). This trend was confirmed by linear regression, suggesting a "major gene" behavior for the T allele. Multiple regression analysis showed that this effect was independent of anthropometric and clinical factors, including adrenal aldosterone. CONCLUSIONS: Our data suggest that -344C/T polymorphism affects LV mass and thickness in essential hypertension, independent of adrenal aldosterone. A role for intracardiac aldosterone synthesis is hypothesized.