Published online before print October 13, 2008, doi: 10.1161/HYPERTENSIONAHA.108.113282
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(Hypertension. 2008;52:980.)
© 2008 American Heart Association, Inc.
Original Articles |
Ambulatory Blood Pressure Is Associated With Polymorphic Variation in P2X Receptor Genes
From the Institute of Human Genetics (J.P.-D., T.J.R., B.K.), School of Mathematics and Statistics (P.J.A.), and Office of the Vice Chancellor (C.R.W.E.), Newcastle University, Newcastle upon Tyne, United Kingdom; Department of Medicine (B.M.M.), University of Cape Town, Cape Town, South Africa; and the Department of Cardiovascular Medicine (M.F., H.W.), University of Oxford, Oxford, United Kingdom.
Correspondence to Bernard Keavney, Institute of Human Genetics, Central Parkway, Newcastle upon Tyne, NE1 3BZ United Kingdom. E-mail b.d.keavney{at}ncl.ac.uk
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Abstract |
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The P2X receptor gene family encodes a series of proteins that function as ATP-gated nonselective ion channels. P2X receptor channels are involved in transducing aldosterone-mediated signaling in the distal renal tubule and are potential candidate genes for blood pressure regulation. We performed a family based quantitative genetic association study in 248 families ascertained through a proband with hypertension to investigate the relationship between common genetic variation in the P2X4, P2X6, and P2X7 genes and ambulatory blood pressure. We genotyped 28 single nucleotide polymorphisms, which together captured the common genetic variability in the 3 genes. We corrected our results for multiple comparisons specifying a false discovery rate of 5%. We found significant evidence of association between the single nucleotide polymorphism rs591874 in the first intron of the P2X7 gene and blood pressure. The strongest association was found for nighttime diastolic blood pressure (P=0.0032), although association was present for both systolic and diastolic blood pressures measured by an observer during the day and at night. Genotypes were associated with a 0.2 SD (
2.5 mm Hg) difference in night diastolic blood pressure per allele and accounted for 1% of the total variability in this measurement. Other suggestive associations were found, but these were nonsignificant after correction for multiple testing. These genetic data suggest that drugs affecting P2X receptor signaling may have promise as clinical antihypertensive agents.
Key Words: blood pressure monitoring • ambulatory • hypertension • receptors • purinergic • purinoceptor P2X6 • purinoceptor P2X4 • purinoceptor P2X7
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Introduction |
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Hypertension is a multifactorial complex disorder involving interactions between genetic and environmental determinants. The heritability of systolic and diastolic blood pressures (BPs) has been estimated at between 30% and 60%.1 Case-control studies of hypertension as a "disease state," a widely used design, introduce an artificial dichotomy into the analysis of a quantitative trait. Even very large case-control genetic studies of hypertension have not, thus far, been successful in identifying susceptibility genes.2 However, BP considered quantitatively presents specific difficulties as a phenotype for study because of the uncertainty in estimating an individual’s "usual" BP from a limited number of measurements. Ambulatory BP measurement has been shown to provide more reproducible estimates of usual BP than isolated clinical readings, and a quantitatively based approach using ambulatory BP measurement, therefore, has the potential to increase the power of genetic studies.3,4
Purinergic P2X receptors are nonselective ATP-gated ion channels that are widely expressed in mammalian cells. Molecular studies have identified 7 P2X receptor genes that form both homotrimeric and heterotrimeric complexes in vivo.5 The P2X4 gene is abundantly expressed in vascular endothelial cells,6 where it is the major mediator of Ca2+ influx induced by ATP and blood flow. P2X4 knockout mice have marked suppression of normal endothelial cell responses to blood flow, have deficient downstream adaptive vascular remodeling, and develop hypertension.7 P2X4 receptors are also expressed in cardiac myocytes8 and in the renal collecting duct.9 Aldosterone stimulates the extracellular release of ATP from the basolateral membrane of renal tubular cells, which acts via P2X4 or P2X4-like receptors to mediate contraction of the adjacent epithelial cells, resulting in transepithelial sodium transport via ENaC.10
The amino acid sequence of P2X7 is closely related to P2X4, and the 2 genes lie adjacent to each other separated by some 24 kb on chromosome 12, suggesting that they may have evolved by a process of gene duplication. The P2X7 gene is expressed in response to injury (eg, diabetic nephropathy) in the podocytes, mesangial cells, and glomerular endothelial cells of the kidney and in vascular endothelial cells. P2X7 is constitutively expressed in the hypothalamus and nucleus tractus solitarius, indicating a potential role for receptors incorporating P2X7 subunits in the regulation of sympathoadrenal tone.
P2X6 receptor subunits coassemble with P2X4 subunits,11 and the P2X6 gene is expressed in the distal renal tubule and the collecting duct principal cells. Some previous genome-wide linkage scans for BP have detected linkage in the region of chromosome 22 that includes the P2X6 gene.12–14 The P2X4, P2X6, and P2X7 genes are, therefore, plausible candidate genes for BP regulation. We have investigated the impact of common variation in these genes on BP in a large, family based association study using ambulatory BP monitoring.
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Methods |
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The collection strategy of this family study has been described previously.15 Families were ascertained through a proband diagnosed with essential hypertension. The following criteria defined eligibility as a proband: a mean systolic BP >140 mm Hg and mean diastolic BP >90 mm Hg on daytime ambulatory BP monitoring, >3 office BP readings >160 mm Hg systolic and 95 mm Hg diastolic, or treatment with
2 antihypertensive drugs. These relatively stringent criteria were applied to provide maximum security that probands were indeed at the upper end of the population BP distribution. Secondary hypertension was excluded using the standard screening protocol applied in the hypertension clinic.
To be suitable for the study, families were required to consist of 3 siblings clinically assessable for BP if 1 parent of the sibship was available to give blood for DNA analysis and to consist of 4 assessable siblings if no parent was available for DNA analysis. Quantitatively assessed sibships were recruited either in the generation of the proband or his or her offspring. Where members of the sibship were found to be hypertensive, families were extended and the spouses and offspring of hypertensive siblings collected. The majority of individuals in the family collection, therefore, had BPs within the conventionally accepted "normal range," and the family collection included some extended families, although most were nuclear families.15 A total of 1425 subjects from 248 families were recruited. The median family size was 5 people, with 60% of families composed of between 4 and 6 genotyped and phenotyped members. A total of 71% of families were 2 generation, and 29% were 3 generation. Eighty-four percent of families had an assessable sibship in the generation of the proband, whereas 16% of families consisted of a proband and their nuclear family (spouse and children >18 years) only. The study received ethical clearance from the appropriate review committees and corresponded with the principles of the Declaration of Helsinki. All of the participants gave informed consent to participate in the study.
BP was measured using ambulatory monitoring for a period of 24 hours in all of the subjects willing to undergo monitoring, using the A&D TM2421 monitor according to a protocol described previously16 (see data supplement available online at http://hyper.ahajournals.org for additional detail). A full clinical history was taken, which included the subject’s medical history and lifestyle factors, including consumption of alcohol and tobacco, as well as habitual physical exercise. Anthropometric measurements, including height, weight, and waist and hip circumferences, were performed. DNA was extracted from blood samples using standard methods.
The P2X4 and P2X7 genes are located adjacent to each other on the long arm of chromosome 12 (12q24.32 and 12q24.2, respectively), and the P2X6 gene is located on chromosome 22 (22q11.21). Tag single nucleotide polymorphisms (SNPs) within each gene and 15 kb in either direction (to incorporate close-range upstream and downstream regulatory sequences) were identified by reference to the SNP data from the HapMap CEU samples of Northern and Western European ancestry (http://www.hapmap.org).17 The tagging strategy was implemented with the Tagger utility in the Haploview software package, with the parameters r2>0.8 and minor allele frequency 0.05.18 14 tag SNPs were required in P2X7, 5 tag SNPs in P2X4, and 6 tag SNPs in P2X6. Selected SNPs, their location in the genes, and linkage disequilibrium between the SNPs (calculated using Haploview from our data) are shown in the Figure.
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Twenty-five SNPs were genotyped by the MassEXTEND method on a Sequenom MassArray matrix assisted laser desorption ionization-time of flight platform. The primers were designed using the MassArray assay designer software (version 3.0.2.0, Sequenom). Details of primers for these assays are shown in Table S1. Three SNPs for which satisfactory Sequenom assays could not be developed were genotyped using restriction fragment length polymorphism assays. DNA sequence for these SNPs was downloaded from the Ensembl database (http://www.ensembl.org)19 and manually analyzed for the presence of restriction sites and to determine primer sequences. PCR primers, annealing temperatures, and restriction enzymes used are shown in Table S2. Control individuals of known genotypes were included in every plate, and 100 randomly selected samples were genotyped twice for each polymorphism. Genotyping was carried out blinded to phenotypic information.
Mendelian inheritance of all of the genotypes and Hardy-Weinberg equilibrium for each marker were checked using PEDSTATS.20 Additional checks based on unlikely recombination patterns within families were carried out using the error-checking option in MERLIN version 1.1.1.21 Errors were corrected when possible by reference to the raw genotyping data, and when this was not possible, genotypes were excluded from analysis. We used MINITAB version 14 to examine the BP phenotypes for normality. All of the BP variables required log-transformation to adequately conform to a normal distribution (see Figure S1). We used MINITAB to adjust the BP phenotypes for the significant covariates age, sex, and habitual physical activity using linear regression. The main analyses included only those individuals in whom BP measurements free from antihypertensive treatment were available, so no adjustment for drug treatment was required. Supplementary analyses including both untreated participants and participants taking antihypertensive medications were performed; in these analyses, the effects of each of the main classes of drugs (diuretics and β-blockers) on BP were estimated from the data by regression and the appropriate adjustment made to the on-treatment BP values. The log-transformed, covariate-adjusted residuals were entered into the quantitative trait genetic association analyses, which were performed (for both untreated-only and all-subject analyses) using a variance-components approach, which takes account of shared polygenic effects in members of the same pedigree, implemented in the QTDT program.22 We first tested for the presence of population stratification for each SNP by comparing the between- and within-family components of variance in QTDT. Because there was no evidence of stratification (P>0.05), we used the total association model, which incorporates both components, in the principal analyses. We also conducted subsidiary analyses using the "orthogonal" or transmission/disequilibrium test model specifiable in QTDT, which tests only transmission from heterozygote parents and, thus, uses only a fraction of the available genotype/phenotype information; despite the considerable loss of power, if such transmission/disequilibrium test analyses are positive, they potentially provide an additional level of security in the result. To take some account of the risk of false-positive findings because of multiple comparisons, we interpreted the results based on the false discovery rate.23 We used the program QVALUE running on top of the statistical package R to determine q values24 (see data supplement). We adopted an arbitrary false discovery rate threshold of 0.05 (ie, 1 in 20 of the associations passing this criterion were expected to be false).
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Results |
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The ambulatory BP measurements in the participants are summarized in Table 1. Fifty-two percent of the participants were women, and 36% were classified as hypertensive. As expected given the selection of families through a hypertensive proband, ambulatory BPs tended to be higher than would be expected in a nonselected population. Quantitative daytime recordings of ambulatory BP were available in 958 people (of whom 126 were pedigree founders), with on-treatment recordings available in a further 212. Because of participants electing to switch off the monitor at night, fewer night recordings were available: quantitative night recordings were available in 770 people (of whom 100 were pedigree founders), with on-treatment recordings available in a further 158. The sibling correlations for the traits ranged from 22% to 36% for the different ambulatory measures and were comparable with previously published family data.25,26
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Genotyping was successful for >98% of the samples for all of the SNPs. The estimated genotype miscall rate was <1% for all of the SNPs. All 25 of the markers were in Hardy-Weinberg equilibrium at the 5% significance level. Table S3 shows genotype frequencies and counts for the population. The minor allele frequencies ranged from 1.9% to 49.0%, yielding marker heterozygosities from 3.6% to 51.0%. Allele frequencies were very similar to the HapMap data for the CEU population.17 As expected from the tagging strategy that we used, the correlation between SNPs was generally modest (Figure) and corresponded well with previous publicly available data. Haplotypes with frequencies of >1% in the population are shown in Tables S4 and S5. Twenty-five such haplotypes were present in the region of chromosome 12 spanning P2X4 and P2X7, accounting for >85% of the genetic variation present. Seventeen such haplotypes were present in the region of chromosome 22 spanning P2X6, accounting for 96% of variation. The composition and frequency of the common haplotypes were in close agreement with those described in the HapMap CEU population.
Using data from participants who were not taking antihypertensive medications, we found significant association between genotypes at the SNP rs591874 in the P2X7 gene and diastolic BP, whether measured by the "clinic readings," during the day or at night (P=0.015, 0.0072, and 0.0032, respectively, among 887, 915, and 742 genotyped and quantitatively phenotyped individuals; QTDT "total" association model; Table 2). Somewhat less significant associations were found between the marker and systolic BP in all 3 of the settings (P=0.028, 0.049, and 0.035, respectively; Table 2). Each rs591874 minor allele was associated with a higher log-transformed, covariate-adjusted diastolic BP as measured by "clinic" readings, daytime and nighttime recordings by 0.126, 0.138, and 0.173 SDs, respectively. In our sample, this would correspond with an increment of between 2 and 3 mm Hg in diastolic BP per allele. Genotype at rs591874 accounted for 1% of the variability in the diastolic BP measurements. Association was also found between the marker rs656612 in the P2X7 gene and nighttime diastolic BP (P=0.0051; among 397 common homozygotes, 295 heterozygotes, and 53 rare homozygotes). At this SNP, each minor allele was associated with a 0.172-SD increase of BP. Genotype at rs656612 accounted for 1.15% of the variability of nighttime diastolic BP. The rs656612 SNP is situated close to rs591874 in intron 1 of the P2X7 gene, and linkage disequilibrium between these 2 SNPs is strong (D’=0.86; r2=0.73); when genotype at rs591874 was included in the model, the effect of rs656612 was no longer significant. Some nominally significant associations (P<0.05) were present for other SNPs (Table S6); however, after correcting for multiple tests using QVALUE, only the associations between rs591874 and rs656612 with diastolic BP remained significant, whereas other associations became nonsignificant at the 5% false discovery rate level. As expected given the loss of approximately half of the genotype/phenotype information entailed in performing the TDT analyses, these did not provide additional strong evidence of association (0.04<P<0.1).
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In supplementary analyses including participants on and off treatment (using identical statistical methodology), the association between the marker rs591874 with day and night diastolic BP became, if anything, slightly more significant (P=0.0028 and 0.00098, respectively, among 1126 and 894 genotyped and phenotyped individuals; Table 2). This was also the case for the association between night diastolic BP and the marker rs656612 (P=0.0016; N=888).
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Discussion |
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We have found association between common variation in the genomic segment including the P2X7 and P2X4 genes and BP. Each minor allele of the markers rs591874 and rs656612 increases diastolic BP by
0.2 SD in a codominant fashion. These associations remain significant after correction for multiple testing. The effects are small, genotypes accounting for only 1% of the trait’s variability. The SNPs rs591874 and rs656612 are both located in intron 1 of the P2X7 gene, and the degree of linkage disequilibrium is high between them (r2=0.75). When both SNPs were included in statistical models, the association appeared principally to be attributable to the rs591874 genotype. Despite the suggestion from previous linkage studies that the region of chromosome 22 harboring the P2X6 receptor may be involved in susceptibility to hypertension, we found no significant associations with SNPs in P2X6 and BP. The association that we observed between rs591874 and BP was strongest for nighttime diastolic BP, although it was nominally significant for both systolic and diastolic BPs, whether measured by the clinic readings, which initiated the 24-hour ambulatory monitoring, by the daytime, or by the nighttime ambulatory readings. Our data, in keeping with some previous studies, show that the heritability of nighttime diastolic ambulatory BP is significantly higher than the other readings, possibly because of the lesser effect of random environmental variables, such as physical activity and mental stressors during the night.27 Our result is, therefore, most likely to reflect the increased power of the study to detect genetic association with the more heritable nighttime diastolic values, despite the fact that the numbers of individuals with quantitative nighttime monitoring values available were significantly lower than the numbers with daytime monitorings or clinic readings. It seems physiologically less likely that our result arises from specific genetic effects on diastolic (as opposed to systolic) and nighttime (as opposed to daytime or clinic) BP, although this cannot be absolutely ruled out. Meta-analysis has shown recently that nighttime BP is a better predictor of adverse outcome than daytime or clinic BP, suggesting that genetic associations with nighttime pressures might be particularly important.28
Because P2X7 and P2X4 lie adjacent to each other on chromosome 12, it is conceivable that the association that we have observed between rs591874 and BP arises through a regulatory effect on either or both of these 2 genes. With respect to P2X7, among the SNPs we typed was rs3751143, which has been shown by others to result in an 50% decrease in receptor function in the heterozygous state and a complete loss of receptor function in the homozygous state.5 We observed no association between BP and this SNP, so it seems unlikely that loss of P2X7 function is the mechanism accounting for our results. We performed in silico analyses to address the possibilities that the associated SNPs, which are intronic, could mediate differences in splicing or modify transcription factor binding sites. Cross-species sequence alignment29 shows quite considerable sequence conservation in intron 1 of P2X7, including the rs591874 SNP, suggesting a potential regulatory role. RESCUE-ESE30 and Automated Splice Site Analysis programs31 show that neither of the SNPs are in consensus splice sequences or generate cryptic splice sites, but the minor allele of the rs591874 SNP abolishes a binding site for the splicing protein SC35. Genetic variants altering SC35 binding sites can have large effects on gene expression.32 Additional studies to quantify the effect of the associated SNPs and of additional SNPs (particularly in the 24-kb regions between the 2 genes, which was not covered with tagSNPs in this study) on the expression of both P2X7 and P2X4 may allow the detection of regulatory effects mediating BP differences.
To our knowledge, this is the first study to systematically examine polymorphic variation in these P2X receptor genes and BP. The relatively large sample size in the present study makes a false-positive result because of random chance less likely. Our focus on quantitative data decreased the potential effect of confounding factors (notably, treatment for hypertension, although inclusion of treated individuals strengthened our result), and the use of ambulatory monitor readings increased our power to detect small genetic effects. Detailed phenotyping enabled us to control for covariates, and our analyses incorporated an adjustment for multiple comparisons. However, this study has certain limitations. As with any novel genetic association, these findings will require replication in other large studies; the effect that we have described is small, so as with certain other genetic associations (eg, PPARG Pro12Ala and type 2 diabetes), the optimal estimate of the effect may be apparent only after several additional studies have been performed. The effect that we show (a change of 2 to 3 mm Hg per allele) is not of sufficient size to be of use in genetic screening for BP risk; future fine-mapping studies will be needed to conclusively identify the SNP or set of SNPs at the P2X7/P2X4 locus that has maximal effect on BP. However, even the most strongly associated SNP in the region, when it is identified, may not have an effect that is of substantial size. Our study does not indicate whether the genetic effect on BP arises from upregulation or downregulation of P2X signaling; future functional studies will be required to determine this with security.
Perspectives
We have found that common genetic variation in the region of the P2X7 and P2X4 genes has a small but significant effect on BP in a white population. The associations seem to follow codominant models and account for 1% of BP variation. Genotypes were associated with differences in diastolic BP of 0.2 SD (2 to 3 mm|Hg) per allele. This is the first clinical study to substantiate previous investigations in laboratory animals and in cell lines that suggest an important role of P2X signaling in BP homeostasis.
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Acknowledgments |
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Sources of Funding
The family collection was funded by the Wellcome Trust. The genotyping was funded by the British Heart Foundation. H.W. and B.K. hold British Heart Foundation Chairs.
Disclosures
None.
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Footnotes |
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The first 2 authors contributed equally to this article.
Received March 18, 2008; first decision April 12, 2008; accepted September 15, 2008.
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