Elaine C. Friel

α-Adducin and Angiotensin I–Converting Enzyme Polymorphisms in Essential Hypertension

This study focused on two genes that have previously been implicated in hypertension and may influence renal sodium handling, adducin, and angiotensin I–converting enzyme (ACE). We compared their polymorphic frequencies and interaction in patients with essential hypertension (n=128) and individually age- and gender-matched normotensive control subjects. The &agr;-adducin G460W polymorphism was genotyped by DNA amplification and restriction digestion. The ACE I/D polymorphism was assayed by a triple-primer method, with a “nested” polymerase chain reaction primer situated completely within the insertion sequence of the I allele. The distributions of genotypes and alleles for the two polymorphisms were not significantly different between the case and control populations, and the cross-classification of cases by &agr;-adducin and ACE genotype gave a distribution similar to that of control subjects. We have previously reported that the distributions of genotypes for two linked polymorphisms in the aldosterone synthase gene (one in the steroidogenic factor-1 [SF-1] binding site and the other an intronic conversion [IC]) were significantly different between this cohort of essential hypertensives and matched control subjects. The cross-classification of cases by &agr;-adducin and SF-1, &agr;-adducin and IC, ACE and SF-1, and ACE and IC genotype gave a distribution similar to that of control subjects. Hence, no evidence was found to suggest an association between either the &agr;-adducin G460W or the ACE I/D polymorphism and hypertension in a careful case-control study. Furthermore, the &agr;-adducin G460W, ACE I/D, and aldosterone synthase SF-1 and IC polymorphisms do not appear to interact in our hypertensive population.

An influence of variation in the aldosterone synthase gene (CYP11B2) on corticosteroid responses to ACTH in normal human subjects.

OBJECTIVE Previous evidence suggests that the efficiency of 11β‐hydroxylase is at least partly heritable and also that it may be mildly impaired in essential hypertension. In both cases, assessment of activity was based on the response of 11‐deoxycorticosterone (DOC) and 11‐deoxycortisol to ACTH. The gene (CYP11B1) coding for this enzyme is highly homologous with and lies a relatively short distance downstream from the gene coding for aldosterone synthase (CYP11B2) on chromosome 8. Two polymorphisms of CYP11B2 have been described. The first involves a change of −344C to T in a putative steroidogenic factor‐1 (SF‐1) binding site and the other, the intron conversion, an exchange of intron 2 for that of CYP11B1. These polymorphisms are in linkage dysequilibrium. Their effects on 11β‐hydroxylation were studied.

Polymorphic Variation in the 11β-Hydroxylase Gene Associates With Reduced 11-Hydroxylase Efficiency

The −344 C/T and intron 2 conversion variants in the CYP11B2 gene, encoding aldosterone synthase, have been associated with markers of impaired 11β-hydroxylase activity. We hypothesize that this association is because of variations in the adjacent 11β-hydroxylase gene (CYP11B1) and arises through linkage disequilibrium between CYP11B1 and CYP11B2. The pattern of variation across the entire CYP11B locus was determined by sequencing 26 normotensive subjects stratified by and homozygous for the −344 and intron conversion variants. Eighty-three variants associated with −344 and intron conversion were identified. Haplotype analysis revealed 4 common haplotypes, accounting for 68% of chromosomes, confirming strong linkage disequilibrium across the region. Two novel CYP11B1 polymorphisms upstream of the coding region (−1889 G/T and −1859 A/G) were identified as contributing to the common haplotypes. Given the potential for such mutations to affect transcriptional regulation of CYP11B1, these were analyzed further. A total of 512 hypertensive subjects from the British Genetics of Hypertension Study population were genotyped for these polymorphisms. A significant association was identified between the −1889 polymorphism and urinary tetrahydrodeoxycortisol/total cortisol metabolite ratio, indicating reduced 11β-hydroxylase efficiency. A similar pattern was observed for the −1859 polymorphism, but this did not achieve statistical significance. Functional studies in vitro using luciferase reporter gene constructs show that these polymorphisms significantly alter the transcriptional response of CYP11B1 to stimulation by adrenocorticotropic hormone or forskolin. This study strongly suggests that the impaired 11β-hydroxylase efficiency associated previously with the CYP11B2 −344 and intron conversion variants is because of linkage with these newly identified polymorphisms in CYP11B1.

Aldosterone synthase gene variation and adrenocortical response to sodium status, angiotensin II and ACTH in normal male subjects

objective  Aldosterone synthase, a key enzyme in the terminal steps of aldosterone synthesis, is encoded by the CYP11B2 gene. A polymorphism in the 5′ coding region of this gene (−344 C/T) is associated with hypertension, particularly with elevation of the aldosterone to renin ratio. A second polymorphism (a conversion in intron 2 to resemble that of the neighbouring 11β‐hydroxylase (CYP11B1) gene) is found in close linkage dysequilibrium with the variant at −344 C/T. The mechanism by which these variants predispose to cardiovascular disease and the precise intermediate phenotype associated with them remains speculative.

Phenotypic consequences of variation across the aldosterone synthase and 11‐beta hydroxylase locus in a hypertensive cohort: data from the MRC BRIGHT Study

Background  Aldosterone is an important cardiovascular hormone; 15% of hypertensive subjects have alteration in aldosterone regulation, defined by a raised ratio of aldosterone to renin (ARR). Studies of the aldosterone synthase gene (CYP11B2) have focused on a single nucleotide polymorphism in the 5′promoter region (–344 C/T). In normotensive subjects, the T allele associates with raised levels of the 11‐deoxysteroids, deoxycorticosterone and 11‐deoxycortisol which are substrates for 11β‐hydroxylase, encoded by the adjacent and homologous gene, CYP11B1. We have speculated that this altered 11β‐hydroxylase efficiency leads to increased ACTH drive to the adrenal gland to maintain cortisol production and reported herein the association between the –344 C/T single nucleotide polymorphism (SNP) and adrenal steroid production in subjects with essential hypertension.

Polymorphic differences from normal in the aldosterone synthase gene (CYP11B2) in patients with primary hyperaldosteronism and adrenal tumour (Conn's syndrome)

OBJECTIVE The hypertension of Conns syndrome is due to autonomous aldosterone production by a unilateral adrenocortical adenoma. The source of tumour initiation and the reasons for excess aldosterone production as opposed to cortisol are not known, although variations in the promoter region of the gene coding for aldosterone synthase (CYP11B2) might account for the altered rate of aldosterone secretion.

Functional effects of genetic variants in the 11β‐hydroxylase (CYP11B1) gene

Objective  We previously described an association between the −344C/T 5′‐untranslated region (UTR) polymorphism in the CYP11B2 (aldosterone synthase) gene and hypertension with a raised aldosterone to renin ratio (ARR); the same genetic variant is also associated with impaired adrenal 11β‐hydroxylase efficiency. The −344 polymorphism does not seem to be functional, so is likely to be in linkage with variants in CYP11B1 that determine the associated variation in 11β‐hydroxylase efficiency. We therefore aimed to determine whether there is an association between CYP11B1 variants and hypertension and/or an altered ARR.

Common Polymorphisms at the CYP17A1 Locus Associate With Steroid Phenotype: Support for Blood Pressure Genome-Wide Association Study Signals at This Locus

Genome-wide association studies implicate the CYP17A1 gene in human blood pressure regulation although the causative polymorphisms are as yet unknown. We sought to identify common polymorphisms likely to explain this association. We sequenced the CYP17A1 locus in 60 normotensive individuals and observed 24 previously identified single-nucleotide polymorphisms with minor allele frequency >0.05. From these, we selected, for further studies, 7 polymorphisms located ≤2 kb upstream of the CYP17A1 transcription start site. In vitro reporter gene assays identified 3 of these (rs138009835, rs2150927, and rs2486758) as having significant functional effects. We then analyzed the association between the 7 polymorphisms and the urinary steroid metabolites in a hypertensive cohort (n=232). Significant associations included that of rs138009835 with aldosterone metabolite excretion; rs2150927 associated with the ratio of tetrahydrodeoxycorticosterone to tetrahydrodeoxycortisol, which we used as an index of 17α-hydroxylation. Linkage analysis showed rs138009835 to be the only 1 of the 7 polymorphisms in strong linkage disequilibrium with the blood pressure–associated polymorphisms identified in the previous studies. In conclusion, we have identified, characterized, and investigated common polymorphisms at the CYP17A1 locus that have functional effects on gene transcription in vitro and associate with corticosteroid phenotype in vivo. Of these, rs138009835—which we associate with changes in aldosterone level—is in strong linkage disequilibrium with polymorphisms linked by genome-wide association studies to blood pressure regulation. This finding clearly has implications for the development of high blood pressure in a large proportion of the population and justifies further investigation of rs138009835 and its effects. # Novelty and Significance {#article-title-30}Genome-wide association studies implicate the CYP17A1 gene in human blood pressure regulation although the causative polymorphisms are as yet unknown. We sought to identify common polymorphisms likely to explain this association. We sequenced the CYP17A1 locus in 60 normotensive individuals and observed 24 previously identified single-nucleotide polymorphisms with minor allele frequency >0.05. From these, we selected, for further studies, 7 polymorphisms located ⩽2 kb upstream of the CYP17A1 transcription start site. In vitro reporter gene assays identified 3 of these (rs138009835, rs2150927, and rs2486758) as having significant functional effects. We then analyzed the association between the 7 polymorphisms and the urinary steroid metabolites in a hypertensive cohort (n=232). Significant associations included that of rs138009835 with aldosterone metabolite excretion; rs2150927 associated with the ratio of tetrahydrodeoxycorticosterone to tetrahydrodeoxycortisol, which we used as an index of 17&agr;-hydroxylation. Linkage analysis showed rs138009835 to be the only 1 of the 7 polymorphisms in strong linkage disequilibrium with the blood pressure–associated polymorphisms identified in the previous studies. In conclusion, we have identified, characterized, and investigated common polymorphisms at the CYP17A1 locus that have functional effects on gene transcription in vitro and associate with corticosteroid phenotype in vivo. Of these, rs138009835—which we associate with changes in aldosterone level—is in strong linkage disequilibrium with polymorphisms linked by genome-wide association studies to blood pressure regulation. This finding clearly has implications for the development of high blood pressure in a large proportion of the population and justifies further investigation of rs138009835 and its effects.

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.

Impaired mitochondrial respiration in human carotid plaque atherosclerosis: A potential role for Pink1 in vascular smooth muscle cell energetics

Background and aims DNA damage and mitochondrial dysfunction are thought to play an essential role in ageing and the energetic decline of vascular smooth muscle cells (VSMCs) essential for maintaining plaque integrity. We aimed to better understand VSMCs and identify potentially useful compensatory pathways that could extend their lifespan. Moreover, we wanted to assess if defects in mitochondrial respiration exist in human atherosclerotic plaques and to identify the appropriate markers that may reflect a switch in VSMC energy metabolism. Methods Human plaque tissue and cells were assessed for composition and evidence of DNA damage, repair capacity and mitochondrial dysfunction. Fresh plaque tissue was evaluated using high resolution oxygen respirometry to assess oxidative metabolism. Recruitment and processing of the mitochondrial regulator of autophagy Pink1 kinase was investigated in combination with transcriptional and protein markers associated with a potential switch to a more glycolytic metabolism. Results Human VSMC have increased nuclear (nDNA) and mitochondrial (mtDNA) damage and reduced repair capacity. A subset of VSMCs within plaque cap had decreased oxidative phosphorylation and expression of Pink1 kinase. Plaque cells demonstrated increased glycolytic activity in response to loss of mitochondrial function. A potential compensatory glycolytic program may act as energetic switch via AMP kinase (AMPK) and hexokinase 2 (Hex2). Conclusions We have identified a subset of plaque VSMCs required for plaque stability that have increased mitochondrial dysfunction and decreased oxidative phosphorylation. Pink1 kinase may initiate a cellular response to promote a compensatory glycolytic program associated with upregulation of AMPK and Hex2.