The major new results of the present study are as follows. First, the SNPs, rs11638442 and rs1045642, in the CYP11A1 and in MDR1 gene, respectively, affect circulating EO. A small set of informative tagSNPs and haplotype analysis for the entire region of these genes in our hypertensive population, which were characterized also for EO levels, allowed us to identify these associations. Haplotype-based analyses of these genes did not increase the power of association with respect to SNP analysis. Second, variants in HSD3B1 are associated with blood pressure and variations in plasma Aldo and potassium among patients with essential hypertension. Third, our investigation provides no evidence for an association of the specific gene variants, including at-risk common haplotypes of the HSD3B2, CYP11A1, and SLCO4C1 genes, with blood pressure or plasma EO in our study population. Nevertheless, under a Bayesian framework with reference to the a priori probability of association, SLCO4C1 and CYP11A1 were associated significantly with DBP and plasma EO levels, respectively. Fourth, the present study shows that plasma EO was significantly related with day and nighttime DBPs among patients with never-treated essential hypertension. This observation reinforces previous reports from our laboratory.
The 3b-hydroxysteroid dehydrogenase/δ5-4 is a key rate-limiting enzyme in steroid biosynthesis pathways that produce estradiol, testosterone, cortisol, and Aldo. It is expressed as two tissue-specific isoforms (HSD3B1 and HSD3B2) with different substrate affinities.30
This enzyme has been shown to be in the biosynthetic pathway for EO based on studies with cultured adrenocortical cells10
and in the MHS genetic model of hypertension.9
We investigated both isoforms considering the following: (i) These two genes are arranged in tandem on the same chromosome; (ii) Although the quantitative analysis in rats identified differential expression for HSD3B2, the oligonucleotide used for gene silencing with RNAi does not distinguish between the two high homologous isoforms;9
(iii) A quantitative trait locus for blood pressure was identified in the HSD3B2 chromosomal region in genome-wide linkage analysis;31
and (iv) In a population of Swedish normotensives, Rosmond et al
found a positive association of blood pressure with HSD3B1 rs6203 (C/T Leu338) with higher SBP and DBP in carrier of the C allele. This SNP was, however, not associated with hypertension in a cohort of Caucasian Australian hypertensives,23
although the study revealed C allele tracked with a higher DBP.23
Our analysis confirms that the C allele is associated with a higher DBP; however, the statistical significance was lost after correction for multiple testing.
TagSNPs and haplotype analysis in our population add further support for the involvement of HSD3B1 in blood pressure regulation, with rs3765945 and rs1047303 showing association with SBP. In contrast, variants in the HSD3B2 gene, located only 84 Kb away from HSD3B1, were not associated with blood pressure in our study. Further tagging of the intergenic region not containing known genes, with two additional informative SNPs, excluded association with the described phenotypes. Furthermore, the observation that the HSD3B1 gene was associated with increased plasma Aldo and lower plasma K+ suggests that a gain of function mutation in HSD3B1 gene in LD with the intronic rs3765945 augments the biosynthesis of this hormone; however, there was no association between these or other SNPs, when both the PRA/Aldo ratio and the urinary K excretion were considered, which seems surprising given that these two parameters may be considered to be a more sensitive index of Aldo action than that measured using the plasma Aldo only.
Functional polymorphism rs1045642 (T/C Ile1145) in the MDR1
gene has been previously identified24,25
as determinant of transporter activity and variability in drug disposition and efficacy. Hoffmeyer et al
showed that individuals homozygous for the T allele had significantly lower intestinal MDR1 and higher plasma levels of digoxin. In contrast, Nakamura et al
and Sakaeda et al
observed higher MDR1 expression in duodenal enterocytes and lower plasma digoxin in association with homozygosity of the T allele. Thus, there is an inverse relationship between MDR expression and serum digoxin levels in humans. In the present study, we restricted our analysis to 4 mutations in the coding region for MDR1 instead of 14 tagSNPs required to tag the entire gene region (http://www.hapmap.org
). We found an association between rs1045642 (T/C Ile1145) and plasma EO with the highest circulating levels in subjects carrying the CC genotype.
It is also known that ouabain, at nanomolar concentrations, can modulate MDR1
gene transcription and PGP synthesis.17
MDR1 is expressed in liver, intestine, kidney, brain, and the adrenal gland and regulates the transport (i.e., intestinal absorption and renal elimination) of many hydrophobic compounds including digoxin34,35
and other steroids. However, ouabain and EO are highly polar steroids; therefore, at present, it seems less likely that PGP would have a significant impact on circulating EO via an excretory pathway. With this in mind, it is of interest that PGP exerts a profound influence on the regulation of the hypothalamic–pituitary–adrenocortical system.34,36
Thus, PGP variants might affect plasma adrenocorticotropic hormone, which is a known stimulator to EO secretion,37
and/or PGP itself may facilitate the transmembrane secretion of EO from the adrenal cortex. Our findings show that functional polymorphisms of the MDR1
gene influence the basal circulating level of EO in hypertensive humans. Further work will be needed to distinguish the mechanism by which MDR genotypes affect plasma EO.
P450scc, the mitochondrial cholesterol side-chain cleavage enzyme, is the only enzyme that catalyzes the conversion of cholesterol to pregnenolone; thus, it is the common pathway leading to the production of progesterone, androgens, estrogen, and other steroid hormones. Moreover, the adrenal biosynthesis of EO is thought to involve cholesterol side-chain cleavage with sequential metabolism of pregnenolone and progesterone.11,37,38
Therefore, it is of interest that we found an association of the CYP11A1
gene with serum cholesterol levels, circulating EO, and DBP in our study population. Plasma cholesterol and high-density lipoprotein-cholesterol are typical multifactorial phenotypes affected by genetic and environmental factors. Accordingly, the influence of a common polymorphism in CYP11A1
genes directly involved in the cholesterol biosynthesis pathway is surprising and suggests, at its simplest level, a substrate-product relationship with EO that will require further studies to define.
Recently, we showed39
that EO in cooperation with the citoskeleton protein adducin, increases tubular Na reabsorption in hypertensives through their action on the Na-K ATPase. Accordingly, we tested the interaction between those genes, involved EO metabolism, and adducin polymorphisms; however, the results were not conclusive because of the small subgroups of patients. Further studies should be carried out in a larger cohort of patients.
In conclusion, among a large cohort of patients with mild-to-moderate hypertension, we employed a candidate-gene approach using a small set of highly informative SNPs to probe for significant associations of blood pressure and related parameters. We observed a significant association of HSD3B1 and CYP11A1 variants with blood pressure, likely based upon contemporary understanding, and to be mediated through plasma Aldo and EO, respectively. Furthermore, we provide the first evidence that variations in the MDR1 gene are linked with plasma EO among patients with never-treated essential hypertension. Our results reinforce the view that inherited variations in steroid biosynthetic pathways as well those potentially involved with EO transmembrane transport have significant effects on the circulating levels of Aldo, EO, and blood pressure in patients with mild-to-moderate essential hypertension.