Timo P. Hiltunen

Genomic Association Analysis of Common Variants Influencing Antihypertensive Response to Hydrochlorothiazide

To identify novel genes influencing blood pressure response to thiazide diuretic therapy for hypertension, we conducted genome-wide association meta-analyses of ≈1.1 million single-nucleotide polymorphisms in a combined sample of 424 European Americans with primary hypertension treated with hydrochlorothiazide from the Pharmacogenomic Evaluation of Antihypertensive Responses study (n=228) and the Genetic Epidemiology of Responses to Antihypertensive study (n=196). Polymorphisms associated with blood pressure response at P<10–5 were tested for replication of the associations in independent samples of hydrochlorothiazide-treated European hypertensives. The rs16960228 polymorphism in protein kinase C, &agr; replicated for same-direction association with diastolic blood pressure response in the Nordic Diltiazem study (n=420) and the Genetics of Drug Responsiveness in Essential Hypertension study (n=206), and the combined 4-study meta-analysis P value achieved genome-wide significance (P=3.3×10−8). Systolic or diastolic blood pressure responses were consistently greater in carriers of the rs16960228 A allele than in GG homozygotes (>4/4 mm Hg) across study samples. The rs2273359 polymorphism in the GNAS-EDN3 region also replicated for same-direction association with systolic blood pressure response in the Nordic Diltiazem study, and the combined 3-study meta-analysis P value approached genome-wide significance (P=5.5×10−8). The findings document clinically important effects of genetic variation at novel loci on blood pressure response to a thiazide diuretic, which may be a basis for individualization of antihypertensive drug therapy and identification of new drug targets.

677 C-->T polymorphism of the methylenetetrahydrofolate reductase gene and preeclampsia.

Objective To evaluate C to T substitution at nucleotide 677 of N5,N10-methylenetetrahydrofolate reductase gene in women with prior preeclamptic or normotensive pregnancies. Methods Methylenetetrahydrofolate reductase genotypes were determined in 113 Finnish women with preeclamptic first pregnancies and 103 controls with one or more normotensive pregnancies, using polymerase chain reaction and restriction enzyme analysis. Preeclampsia was defined as severe in 100 women who fulfilled one or more of the subsequent criteria: systolic blood pressure (BP) at least 160 mmHg, diastolic BP at least 110 mmHg, or proteinuria at least 2 g per 24-hour urine collection. Results There were no significant differences in prevalences of the methylenetetrahydrofolate reductase genotypes (CC, CT, and TT) between groups (57%, 40%, and 3% in the preeclamptic group and 54%, 39%, and 7%, respectively, in controls). The frequency of the T677 allele was 0.23 in the preeclamptic group and 0.26 in the control group (difference 0.03; 95% confidence interval −0.08, 0.14; P = .51). Our sample had 60% power to detect a difference of the allele frequencies similar to that (0.12) reported previously. The result was similar when analysis was restricted to patients with severe preeclampsia (T677 allele frequency 0.22). Conclusion A carrier status for the T677 allele of the methylenetetrahydrofolate reductase gene does not predispose to preeclampsia, at least in the Finnish population.

Common variants of the beta and gamma subunits of the epithelial sodium channel and their relation to plasma renin and aldosterone levels in essential hypertension

BackgroundRare mutations of the epithelial sodium channel (ENaC) result in the monogenic hypertension form of Liddles syndrome. We decided to screen for common variants in the ENaC βand γ subunits in patients with essential hypertension and to relate their occurrence to the activity of circulating renin-angiotensin-aldosterone system.MethodsInitially, DNA samples from 27 patients with low renin/low aldosterone hypertension were examined. The DNA variants were subsequently screened for in 347 patients with treatment-resistant hypertension, 175 male subjects with documented long-lasting normotension and 301 healthy Plasma renin and aldosterone levels were measured under baseline conditions and during postural and captopril challenge tests.ResultsTwo commonly occurring βENaC variants (G589S and a novel intronic i12-17CT substitution) and one novel γENaC variant (V546I) were detected. One of these variants occurred in a heterozygous form in 32 patients, a prevalence (9.2%) significantly higher than that in normotensive males (2.9%, p = 0.007) and blood donors (3.0%, p = 0.001). βENaC i12-17CT was significantly more prevalent in the hypertension group than in the two control groups combined (4.6% vs. 1.1%, p = 0.001). When expressed in Xenopus oocytes, neither of the two ENaC amino acid-changing variants showed a significant difference in activity compared with ENaC wild-type. No direct evidence for a mRNA splicing defect could be obtained for the βENaC intronic variant. The ratio of daily urinary potassium excretion to upright and mean (of supine and upright values) plasma renin activity was higher in variant allele carriers than in non-carriers (p = 0.034 and p = 0.048).ConclusionsAt least 9% of Finnish patients with hypertension admitted to a specialized center carry genetic variants of β and γENaC, a three times higher prevalence than in the normotensive individuals or in random healthy controls. Patients with the variant alleles showed an increased urinary potassium excretion rate in relation to their renin levels.

Pharmacogenomics of Hypertension: A Genome‐Wide, Placebo‐Controlled Cross‐Over Study, Using Four Classes of Antihypertensive Drugs

Background Identification of genetic markers of antihypertensive drug responses could assist in individualization of hypertension treatment. Methods and Results We conducted a genome‐wide association study to identify gene loci influencing the responsiveness of 228 male patients to 4 classes of antihypertensive drugs. The Genetics of Drug Responsiveness in Essential Hypertension (GENRES) study is a double‐blind, placebo‐controlled cross‐over study where each subject received amlodipine, bisoprolol, hydrochlorothiazide, and losartan, each as a monotherapy, in a randomized order. Replication analyses were performed in 4 studies with patients of European ancestry (PEAR Study, N=386; GERA I and II Studies, N=196 and N=198; SOPHIA Study, N=372). We identified 3 single‐nucleotide polymorphisms within the ACY3 gene that showed associations with bisoprolol response reaching genome‐wide significance (P<5×10−8); however, this could not be replicated in the PEAR Study using atenolol. In addition, 39 single‐nucleotide polymorphisms showed P values of 10−5 to 10−7. The 20 top‐associated single‐nucleotide polymorphisms were different for each antihypertensive drug. None of these top single‐nucleotide polymorphisms co‐localized with the panel of >40 genes identified in genome‐wide association studies of hypertension. Replication analyses of GENRES results provided suggestive evidence for a missense variant (rs3814995) in the NPHS1 (nephrin) gene influencing losartan response, and for 2 variants influencing hydrochlorothiazide response, located within or close to the ALDH1A3 (rs3825926) and CLIC5 (rs321329) genes. Conclusions These data provide some evidence for a link between biology of the glomerular protein nephrin and antihypertensive action of angiotensin receptor antagonists and encourage additional studies on aldehyde dehydrogenase–mediated reactions in antihypertensive drug action.

Laboratory tests as predictors of the antihypertensive effects of amlodipine, bisoprolol, hydrochlorothiazide and losartan in men: results from the randomized, double-blind, crossover Genres Study

Objective Individual blood pressure responses to antihypertensive therapy are difficult to predict. To improve optimization of antihypertensive therapy, we analyzed correlations of relevant laboratory tests with blood pressure responses to four antihypertensive monotherapies. Methods In the GENRES study, 208 Finnish men aged 35–60 years with moderate hypertension used amlodipine 5 mg, bisoprolol 5 mg, hydrochlorothiazide 25 mg and losartan 50 mg daily, each for 4 weeks as a monotherapy in a double-blind, randomized, placebo-controlled crossover study; that is, each subject received each type of monotherapy in a random order. The treatment periods were preceded and separated by 4-week placebo periods. Ambulatory 24-h and office blood pressure measurements were carried out after all study periods. Data from several biochemical tests were correlated to antihypertensive drug responses. Results Serum total calcium concentration was negatively correlated with blood pressure responses to amlodipine (P values 0.001–0.002). Plasma renin activity was positively correlated with blood pressure responses to losartan (P values 0.001–0.005) and bisoprolol (P values 0.03–0.17), and negatively with blood pressure responses to hydrochlorothiazide (P values 0.01–0.07). Daily urinary excretion of sodium was negatively correlated with ambulatory blood pressure responses to amlodipine (P values 0.001–0.01). Conclusions In this carefully controlled study, marked individual variations in antihypertensive drug responsiveness were found to correlate to several baseline laboratory parameters. The negative correlation between serum calcium levels and amlodipine responses is intriguing and suggests an underlying mechanistic association. Collectively, our data imply that laboratory tests may have some value in prediction of the efficacy of various antihypertensive drug therapies, although great patient-to-patient variation remains an obstacle for exact predictive classification.

Relationship of electrocardiographic repolarization measures to echocardiographic left ventricular mass in men with hypertension.

Objective Arterial hypertension often leads to an increase in left ventricular mass (LVM). Marked left ventricular hypertrophy (LVH) is associated with potentially arrhythmogenic ventricular repolarization abnormalities, which may contribute to the increased risk of sudden cardiac death in this disorder. We studied whether electrocardiographic repolarization changes are already detectable in mild LVM increase associated with hypertension. Methods In 220 men (mean age 51 ± 6 years) attending the GENRES hypertension study, we measured QT intervals (QTend and QTpeak), T-wave peak to T-wave end (TPE) intervals, and novel T-wave morphology parameters (principal component analysis ratio, T-wave morphology dispersion, total cosine R-to-T, and T-wave residuum) from a digital standard 12-lead electrocardiogram, and related them to echocardiographically determined LVM. Results In this group of moderately hypertensive men, the mean LVM index (LVMI; LVM divided by body surface area) was 99 ± 19 g/m2, with only 18% of the subjects showing evidence of echocardiographic LVH (LVMI > 116 g/m2). LVMI correlated significantly with QT intervals (r = 0.16–0.21, P = 0.018–0.002), TPE intervals (r = 0.23–0.27, P < 0.001), and T-wave morphology parameters (r = 0.22–0.39, P < 0.001). Except for the QTpeak interval, the relationship between LVMI and electrocardiographic repolarization parameters was independent in multivariate analyses. Conclusion Altered electrocardiographic ventricular repolarization, indicating reduced repolarization reserve and possibly increased repolarization heterogeneity, is already present in hypertensive men with only mild LVM increase. At a population level, this may carry important risk implications for the large group of hypertensive patients.

Common genetic variation of β1- and β2-adrenergic receptor and response to four classes of antihypertensive treatment

Varying results have been reported on the association of β-adrenergic receptor polymorphisms with blood pressure (BP) response to β-blockers. We investigated the influence of ADRB1 Ser49Gly and Arg389Gly, and ADRB2 Gly16Arg and Glu27Gln polymorphisms on ambulatory BP response to bisoprolol and three other antihypertensive drug monotherapies in a placebo-controlled, double-blind, cross-over study with 233 moderately hypertensive men. ADRB1 Ser49Ser homozygotes tended to have a better ambulatory BP response to bisoprolol but the difference was statistically nonsignificant. ADRB1 Arg389Arg homozygotes did not show better BP response to bisoprolol than the other genotypes. There were no significant associations of ADRB2 polymorphisms with BP responses to any of the study drugs. The results from this controlled study in hypertensive men do not support clinical use of common polymorphisms in ADRB1 and ADRB2 in predicting BP responses to β-blockers or to three other antihypertensive drugs.

Renin–Angiotensin System and α-Adducin Gene Polymorphisms and Their Relation to Responses to Antihypertensive Drugs: Results From the GENRES Study

BACKGROUND Polymorphisms in genes coding for components of the renin-angiotensin system (RAS) and alpha-adducin (ADD1) have been reported to be associated with blood pressure (BP) responses to antihypertensive agents. The results, however, have not been consistent and most of the earlier studies have been small and lacked placebo-control. Therefore, the association of common polymorphisms in these genes with BP responses to four different antihypertensive drugs was analyzed in a controlled study. METHODS The study included 208 hypertensive Finnish men from the GENRES study. All of them used amlodipine 5 mg, bisoprolol 5 mg, hydrochlorothiazide (HCT) 25 mg, and losartan 50 mg daily, each for 4 weeks as a monotherapy in a double-blind, randomized, study. The treatment periods were separated by 4-week placebo periods. Both 24-h ambulatory (ABP) and office BP (OBP) measurements were carried out. The polymorphisms analyzed were ADD1 Gly460Trp, angiotensinogen (AGT) Met235Thr, angiotensin converting enzyme (ACE) insertion/deletion (I/D), and angiotensin II type 1 receptor (AGTR1) 1166A/C. RESULTS The presence of 460Trp allele of ADD1, previously suggested to be a marker of thiazide responsiveness, did not predict a better response to HCT. There was no significant association of AGT Met235Thr, ACE I/D, and AGTR1 1166A/C polymorphisms with BP responses to the study drugs. ADD1 460Trp and AGT 235Thr alleles were associated with higher systolic white coat effect (WCE) during the placebo periods (P values 0.03 and 0.01, respectively). CONCLUSIONS Common polymorphisms of ADD1, AGT, ACE, and AGTR1 do not markedly predict BP responses to amlodipine, bisoprolol, HCT, and losartan, at least in white hypertensive men.

Genome-Wide and Gene-Based Meta-Analyses Identify Novel Loci Influencing Blood Pressure Response to Hydrochlorothiazide

This study aimed to identify novel loci influencing the antihypertensive response to hydrochlorothiazide monotherapy. A genome-wide meta-analysis of blood pressure (BP) response to hydrochlorothiazide was performed in 1739 white hypertensives from 6 clinical trials within the International Consortium for Antihypertensive Pharmacogenomics Studies, making it the largest study to date of its kind. No signals reached genome-wide significance (P<5×10−8), and the suggestive regions (P<10−5) were cross-validated in 2 black cohorts treated with hydrochlorothiazide. In addition, a gene-based analysis was performed on candidate genes with previous evidence of involvement in diuretic response, in BP regulation, or in hypertension susceptibility. Using the genome-wide meta-analysis approach, with validation in blacks, we identified 2 suggestive regulatory regions linked to gap junction protein &agr;1 gene (GJA1) and forkhead box A1 gene (FOXA1), relevant for cardiovascular and kidney function. With the gene-based approach, we identified hydroxy-delta-5-steroid dehydrogenase, 3 &bgr;- and steroid &dgr;-isomerase 1 gene (HSD3B1) as significantly associated with BP response (P<2.28×10−4). HSD3B1 encodes the 3&bgr;-hydroxysteroid dehydrogenase enzyme and plays a crucial role in the biosynthesis of aldosterone and endogenous ouabain. By amassing all of the available pharmacogenomic studies of BP response to hydrochlorothiazide, and using 2 different analytic approaches, we identified 3 novel loci influencing BP response to hydrochlorothiazide. The gene-based analysis, never before applied to pharmacogenomics of antihypertensive drugs to our knowledge, provided a powerful strategy to identify a locus of interest, which was not identified in the genome-wide meta-analysis because of high allelic heterogeneity. These data pave the way for future investigations on new pathways and drug targets to enhance the current understanding of personalized antihypertensive treatment.

Genome-wide association study identifies CAMKID variants involved in blood pressure response to losartan: the SOPHIA study

BACKGROUND Essential hypertension arises from the combined effect of genetic and environmental factors. A pharmacogenomics approach could help to identify additional molecular mechanisms involved in its pathogenesis. AIM The aim of SOPHIA study was to identify genetic polymorphisms regulating blood pressure response to the angiotensin II receptor blocker, losartan, with a whole-genome approach. MATERIALS & METHODS We performed a genome-wide association study on blood pressure response in 372 hypertensives treated with losartan and we looked for replication in two independent samples. RESULTS We identified a peak of association in CAMK1D gene (rs10752271, effect size -5.5 ± 0.94 mmHg, p = 1.2 × 10(-8)). CAMK1D encodes a protein that belongs to the regulatory pathway involved in aldosterone synthesis. We tested the specificity of rs10752271 for losartan in hypertensives treated with hydrochlorothiazide and we validated it in silico in the GENRES cohort. CONCLUSION Using a genome-wide approach, we identified the CAMK1D gene as a novel locus associated with blood pressure response to losartan. CAMK1D gene characterization may represent a useful tool to personalize the treatment of essential hypertension.