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1.  Association of variants in NEDD4L with blood pressure response and adverse cardiovascular outcomes in hypertensive patients treated with thiazide diuretics 
Journal of hypertension  2013;31(4):698-704.
Objective
Single-nucleotide polymorphisms (SNPs) in NEDD4L may influence the ability of the NEDD4L protein to reduce epithelial sodium channel expression. A variant in NEDD4L, rs4149601, was associated with antihypertensive response and cardiovascular outcomes during treatment with thiazide diuretics and β-blockers in a Swedish population. We sought to further evaluate associations between NEDD4L polymorphisms, blood pressure response and cardiovascular outcomes with thiazide diuretics and β-blockers.
Methods
Four SNPs, rs4149601, rs292449, rs1008899 and rs75982813, were genotyped in 767 patients from the Pharmacogenomic Evaluation of Antihypertensive Responses (PEAR) clinical trial and association was assessed with blood pressure response to hydrochlorothiazide and atenolol. One SNP, rs4149601, was also genotyped in 1345 patients from the International Verapmil SR Trandolapril Study (INVEST), and association was examined with adverse cardiovascular outcomes relative to hydrochlorothiazide treatment.
Results
Significant associations or trends were found between rs4149601, rs292449, rs75982813 and rs1008899 and decreases in blood pressure in whites on hydrochlorothiazide, and a significant association was observed with increasing copies of the GC rs4149601-rs292449 haplotype and greater blood pressure response to hydrochlorothiazide in whites (P = 0.0006 and 0.006, SBP and DBP, respectively). Significant associations were also seen with rs4149601 and an increased risk for adverse cardiovascular outcomes in whites not treated with hydrochlorothiazide [P = 0.022, odds ratio (95% confidence interval) = 10.65 (1.18–96.25)].
Conclusion
NEDD4L rs4149601, rs292449 and rs75982813 may be predictors for blood pressure response to hydrochlorothiazide in whites, and NEDD4L rs4149601 may be a predictor for adverse cardiovascular outcomes in whites not treated with hydrochlorothiazide.
doi:10.1097/HJH.0b013e32835e2a71
PMCID: PMC3756535  PMID: 23353631
epithelial sodium channel; hypertension; International Verapamil SR Trandolapril Study; neural precursor cell expressed developmentally down-regulated 4 like; Pharmacogenomic Evaluation of Antihypertensive Responses; pharmacogenetics
2.  Hypertension Susceptibility Loci and Blood Pressure Response to Antihypertensives – Results from the Pharmacogenomic Evaluation of Antihypertensive Responses (PEAR) Study 
Background
To date, 39 SNPs have been associated with blood pressure (BP) or hypertension (HTN) in genome-wide association studies (GWAS) in Caucasians. Our hypothesis is that the loci/SNPs associated with BP/HTN are also associated with BP response to antihypertensive drugs.
Methods and Results
We assessed the association of these loci with BP response to atenolol or hydrochlorothiazide monotherapy in 768 hypertensive participants in the Pharmacogenomics Responses of Antihypertensive Responses (PEAR) study. Linear regression analysis was performed in Caucasians for each SNP in an additive model adjusting for baseline BP, age, gender and principal components for ancestry. Genetic scores were constructed to include SNPs with nominal associations and empirical p values were determined by permutation test. Genotypes of 37 loci were obtained from Illumina 50K cardiovascular or Omni1M GWAS chips. In Caucasians, no SNPs reached Bonferroni-corrected alpha of 0.0014, six reached nominal significance (p<0.05) and 3 were associated with atenolol BP response at p < 0.01. The genetic score of the atenolol BP lowering alleles was associated with response to atenolol (p =3.3*10−6 for SBP; p=1.6*10−6 for DBP). The genetic score of the HCTZ BP lowering alleles was associated with response to HCTZ (p = 0.0006 for SBP; p = 0.0003 for DBP). Both risk score p values were < 0.01 based on the empirical distribution from the permutation test.
Conclusions
These findings suggest selected signals from hypertension GWAS may predict BP response to atenolol and HCTZ when assessed through risk scoring.
doi:10.1161/CIRCGENETICS.112.964080
PMCID: PMC3529147  PMID: 23087401
beta-blocker; diuretics; hypertension; pharmacogenetics; polymorphisms blood pressure
3.  Association of Chromosome 12 locus with antihypertensive response to hydrochlorothiazide may involve differential YEATS4 expression 
The pharmacogenomics journal  2012;13(3):257-263.
A recent genome-wide analysis discovered an association between a haplotype (from rs317689/rs315135/rs7297610) on Chromosome 12q15 and blood pressure response to hydrochlorothiazide in African-Americans. Our aim was to replicate this association and investigate possible functional mechanisms. We observed similar associations between this haplotype and hydrochlorothiazide response in an independent sample of 746 Caucasians and African-Americans randomized to hydrochlorothiazide or atenolol treatment. The haplotype association was driven by variation at rs7297610, where C/C genotypes were associated with greater mean (systolic: 3.4mmHg, P=0.0275; diastolic: 2.5mmHg, P=0.0196) responses to hydrochlorothiazide vs. T-allele carriers. Such an association was absent in atenolol-treated participants, supporting this as hydrochlorothiazide-specific. Expression analyses in hydrochlorothiazide-treated African-Americans showed differential leukocyte YEATS4 expression between rs7297610 genotype groups at baseline (P=0.024), and reduced expression in C/C genotypes (P=0.009), but not in T-carriers. Our data confirm previous genome-wide findings at 12q15 and suggest differential YEATS4 expression could underpin rs7297610-associated HCTZ response variability, which may have future implications for guiding thiazide treatment.
doi:10.1038/tpj.2012.4
PMCID: PMC3360116  PMID: 22350108
hydrochlorothiazide; hypertension; pharmacogenomics; blood pressure; YEATS4; diuretics
4.  Atenolol Induced HDL-C Change in the Pharmacogenomic Evaluation of Antihypertensive Responses (PEAR) Study 
PLoS ONE  2013;8(10):e76984.
We sought to identify novel pharmacogenomic markers for HDL-C response to atenolol in participants with mild to moderate hypertension. We genotyped 768 hypertensive participants from the Pharmacogenomic Evaluation of Antihypertensive Responses (PEAR) study on the Illumina HumanCVD Beadchip. During PEAR, participants were randomized to receive atenolol or hydrochlorothiazide. Blood pressure and cholesterol levels were evaluated at baseline and after treatment. This study focused on participants treated with atenolol monotherapy. Association with atenolol induced HDL-C change was evaluated in 232 whites and 152 African Americans using linear regression. No SNPs achieved a Bonferroni corrected P-value. However, we identified 13 regions with consistent association across whites and African Americans. The most interesting of these regions were seven with prior associations with HDL-C, other metabolic traits, or functional implications in the lipid pathway: GALNT2, FTO, ABCB1, LRP5, STARD3NL, ESR1, and LIPC. Examples are rs2144300 in GALNT2 in whites (P=2.29x10-4, β=-1.85 mg/dL) and rs12595985 in FTO in African Americans (P=2.90x10-4, β=4.52 mg/dL), both with consistent regional association (P<0.05) in the other race group. Additionally, baseline GALNT2 expression differed by rs2144300 genotype in whites (P=0.0279). In conclusion, we identified multiple gene regions associated with atenolol induced HDL-C change that were consistent across race groups, several with functional implications or prior associations with HDL-C.
doi:10.1371/journal.pone.0076984
PMCID: PMC3792156  PMID: 24116192
5.  G PROTEIN RECEPTOR KINASE 4 (GRK4) POLYMORPHISMS: BETA-BLOCKER PHARMACOGENETICS AND TREATMENT RELATED OUTCOMES IN HYPERTENSION 
Hypertension  2012;60(4):957-964.
G protein-coupled receptor kinases (GRKs) are important regulatory proteins for many G protein-coupled receptors, but little is known about GRK4 pharmacogenetics. We hypothesized three nonsynonymous GRK4 SNPs, R65L (rs2960306), A142V (rs1024323) and A486V (rs1801058) would be associated with blood pressure response to atenolol, but not hydrochlorothiazide, and would be associated with long term cardiovascular outcomes (all cause, death, nonfatal myocardial infarction, nonfatal stroke) in participants treated with an atenolol-based versus verapamil-SR-based antihypertensive strategy. GRK4 SNPs were genotyped in 768 hypertensive participants from the Pharmacogenomic Evaluation of Antihypertensive Responses (PEAR) trial. In Caucasians and African Americans, increasing copies of the variant 65L-142V haplotype were associated with significantly reduced atenolol-induced diastolic blood pressure lowering (−9.1 ± 6.8 vs −6.8 ± 7.1 vs −5.3 ± 6.4 mmHg in participants with 0, 1 and 2 copies of 65L-142V respectively; p=0.0088). 1460 participants with hypertension and coronary artery disease from the INternational VErapamil SR / Trandolapril STudy (INVEST) were genotyped and variant alleles of all three GRK4 SNPs were associated with increased risk for adverse cardiovascular outcomes in an additive fashion, with 486V homozygotes reaching statistical significance (Odds ratio 2.29 [1.48–3.55], p=0.0002). These effects on adverse cardiovascular outcomes were independent of antihypertensive treatment. These results suggest the presence of GRK4 variant alleles may be important determinants of blood pressure response to atenolol and risk for adverse cardiovascular events. The associations with GRK4 variant alleles were stronger in patients who were also ADRB1 389R-homozygotes, suggesting a potential interaction between these two genes.
doi:10.1161/HYPERTENSIONAHA.112.198721
PMCID: PMC3462355  PMID: 22949529
hypertension; GRK4; atenolol; beta-blocker; outcomes; ADRB1; pharmacogenetics
6.  Genetic variants associated with warfarin dose in African-American individuals: a genome-wide association study 
Lancet  2013;382(9894):790-796.
Summary
Background
VKORC1 and CYP2C9 are important contributors to warfarin dose variability, but explain less variability for individuals of African descent than for those of European or Asian descent. We aimed to identify additional variants contributing to warfarin dose requirements in African Americans.
Methods
We did a genome-wide association study of discovery and replication cohorts. Samples from African-American adults (aged ≥18 years) who were taking a stable maintenance dose of warfarin were obtained at International Warfarin Pharmacogenetics Consortium (IWPC) sites and the University of Alabama at Birmingham (Birmingham, AL, USA). Patients enrolled at IWPC sites but who were not used for discovery made up the independent replication cohort. All participants were genotyped. We did a stepwise conditional analysis, conditioning first for VKORC1 −1639G→A, followed by the composite genotype of CYP2C9*2 and CYP2C9*3. We prespecified a genome-wide significance threshold of p<5×10−8 in the discovery cohort and p<0·0038 in the replication cohort.
Findings
The discovery cohort contained 533 participants and the replication cohort 432 participants. After the prespecified conditioning in the discovery cohort, we identified an association between a novel single nucleotide polymorphism in the CYP2C cluster on chromosome 10 (rs12777823) and warfarin dose requirement that reached genome-wide significance (p=1·51×10−8). This association was confirmed in the replication cohort (p=5·04×10−5); analysis of the two cohorts together produced a p value of 4·5×10−12. Individuals heterozygous for the rs12777823 A allele need a dose reduction of 6·92 mg/week and those homozygous 9·34 mg/week. Regression analysis showed that the inclusion of rs12777823 significantly improves warfarin dose variability explained by the IWPC dosing algorithm (21% relative improvement).
Interpretation
A novel CYP2C single nucleotide polymorphism exerts a clinically relevant effect on warfarin dose in African Americans, independent of CYP2C9*2 and CYP2C9*3. Incorporation of this variant into pharmacogenetic dosing algorithms could improve warfarin dose prediction in this population.
Funding
National Institutes of Health, American Heart Association, Howard Hughes Medical Institute, Wisconsin Network for Health Research, and the Wellcome Trust.
doi:10.1016/S0140-6736(13)60681-9
PMCID: PMC3759580  PMID: 23755828
7.  Lack of association of the HMGA1 IVS5-13insC variant with type 2 diabetes in an ethnically diverse hypertensive case control cohort 
Background
Recently, the high-mobility group A1 gene (HMGA1) variant IVS5-13insC has been associated with type 2 diabetes, but reported associations are inconsistent and data are lacking in Hispanic and African American populations. We sought to investigate the HMGA1-diabetes association and to characterize IVS5-13insC allele frequencies and linkage disequilibrium (LD) in 3,070 Caucasian, Hispanic, and African American patients from the INternational VErapamil SR-Trandolapril STudy (INVEST).
Methods
INVEST was a randomized, multicenter trial comparing two antihypertensive treatment strategies in an ethnically diverse cohort of hypertensive, coronary artery disease patients. Controls, who were diabetes-free throughout the study, and type 2 diabetes cases, either prevalent or incident, were genotyped for IVS5-13insC using Taqman®, confirmed with Pyrosequencing and Sanger sequencing. For LD analysis, genotyping for eight additional HMGA1 single nucleotide polymorphisms (SNPs) was performed using the Illumina® HumanCVD BeadChip. We used logistic regression to test association of the HMGA1 IVS5-13insC and diabetes, adjusted for age, gender, body mass index, and percentage European, African, and Native American ancestry.
Results
We observed IVS5-13insC minor allele frequencies consistent with previous literature in Caucasians and African Americans (0.03 in cases and 0.04 in controls for both race/ethnic groups), and higher frequencies in Hispanics (0.07 in cases and 0.07 in controls). The IVS5-13insC was not associated with type 2 diabetes overall (odds ratio 0.98 [0.76-1.26], p=0.88) or in any race/ethnic group. Pairwise LD (r2) of IVS5-13insC and rs9394200, a SNP previously used as a tag SNP for IVS5-13insC, was low (r2=0.47 in Caucasians, r2=0.25 in Hispanics, and r2=0.06 in African Americans). Furthermore, in silico analysis suggested a lack of functional consequences for the IVS5-13insC variant.
Conclusions
Our results suggest that IVS5-13insC is not a functional variant and not associated with type 2 diabetes in an ethnically diverse, hypertensive, coronary artery disease population. Larger, more adequately powered studies need to be performed to confirm our findings.
Trial registration
clinicaltrials.gov (NCT00133692)
doi:10.1186/1479-5876-11-12
PMCID: PMC3558451  PMID: 23302499
HMGA1; Type 2 diabetes; Genetics
8.  CXCL5 polymorphisms are associated with variable blood pressure in cardiovascular disease-free adults 
Human Genomics  2012;6(1):9.
Objective
Leukocyte count has been associated with blood pressure, hypertension, and hypertensive complications. We hypothesized that polymorphisms in the CXCL5 gene, which encodes the neutrophilic chemokine ENA-78, are associated with blood pressure in cardiovascular disease (CVD)-free adults and that these polymorphisms are functional.
Methods and results
A total of 192 community-dwelling participants without CVD or risk equivalents were enrolled. Two CXCL5 polymorphisms (−156 G > C (rs352046) and 398 G > A (rs425535)) were tested for associations with blood pressure. Allele-specific mRNA expression in leukocytes was also measured to determine whether heterozygosity was associated with allelic expression imbalance. In −156 C variant carriers, systolic blood pressure (SBP) was 7 mmHg higher than in −156 G/G wild-type homozygotes (131 ± 17 vs. 124 ± 14 mmHg; P = 0.008). Similarly, diastolic blood pressure (DBP) was 4 mmHg higher in −156 C variant carriers (78 ± 11 vs. 74 ± 11 mmHg; P = 0.013). In multivariate analysis of SBP, age, sex, body mass index, and the −156 G > C polymorphism were identified as significant variables. Age, sex, and the −156 G > C SNP were further associated with DBP, along with white blood cells. Allelic expression imbalance and significantly higher circulating ENA-78 concentrations were noted for variant carriers.
Conclusion
CXCL5 gene polymorphisms are functional and associated with variable blood pressure in CVD-free individuals. The role of CXCL5 as a hypertension- and CVD-susceptibility gene should be further explored.
doi:10.1186/1479-7364-6-9
PMCID: PMC3505480  PMID: 23245743
CXCL5; ENA-78; Blood pressure; Hypertension; Leukocytes
9.  Chromosome 9p21 Haplotypes and Prognosis in Caucasian and African American Patients with Coronary Artery Disease 
Background
While numerous SNPs in Chromosome 9p21 have been associated with coronary artery disease (CAD) and incident MI in Caucasians, there are limited and conflicting reports on the association of this locus with prognosis in Caucasians with existing CAD and no reports in blacks or Hispanics. We investigated the hypothesis that 9p21 polymorphisms are associated with increased risk for adverse cardiovascular outcomes in patients with documented CAD.
Methods and Results
We studied the association of 155 chromosome 9p21 SNPs with adverse outcomes among hypertensive CAD patients of multiple races/ethnicities in INVEST GENES (the INternational VErapamil SR Trandolapril STudy GENetic Substudy, n= 1,460, n = 5,979 for 2 SNPs) and with replication testing of 4 SNPs in the INFORM (INvestigation oF Outcomes from acute coronary syndRoMe; n=714) study of acute coronary syndrome (ACS) patients. In INVEST, the haplotype comprised of the risk allele for the widely reported 9p21 SNPs was associated with better prognosis in Caucasians (OR, 95% CI: 0.72, 0.57–0.92, p = 0.0085) but not blacks (1.21, 0.68–1.24, p = 0.52) or Hispanics (0.96, 0.65–1.44, p=0.86). A less commonly reported LD block was associated with worse prognosis in INVEST among both Caucasians (OR=1.52 (1.20–1.93), p = 0.0006) and African Americans (OR = 4.11 (1.55–10.88), p = 0.004).
Conclusions
Our findings suggest previously reported chromosome 9p21 SNPs, which predict incident CAD, are not associated with higher risk for adverse outcomes in patients with established CAD. The less commonly reported LD block warrants further investigation.
doi:10.1161/CIRCGENETICS.110.959296
PMCID: PMC3101633  PMID: 21372283
chromosome 9p21; cardiovascular outcomes; genetic polymorphisms; INVEST; INFORM
10.  Effects of genetic variation in H3K79 methylation regulatory genes on clinical blood pressure and blood pressure response to hydrochlorothiazide 
Background
Nearly one-third of the United States adult population suffers from hypertension. Hydrochlorothiazide (HCTZ), one of the most commonly used medications to treat hypertension, has variable efficacy. The renal epithelial sodium channel (ENaC) provides a mechanism for fine-tuning sodium excretion, and is a major regulator of blood pressure homeostasis. DOT1L, MLLT3, SIRT1, and SGK1 encode genes in a pathway that controls methylation of the histone H3 globular domain at lysine 79 (H3K79), thereby modulating expression of the ENaCα subunit. This study aimed to determine the role of variation in these regulatory genes on blood pressure response to HCTZ, and secondarily, untreated blood pressure.
Methods
We investigated associations between genetic variations in this candidate pathway and HCTZ blood pressure response in two separate hypertensive cohorts (clinicaltrials.gov NCT00246519 and NCT00005520). In a secondary, exploratory analysis, we measured associations between these same genetic variations and untreated blood pressure. Associations were measured by linear regression, with only associations with P ≤ 0.01 in one cohort and replication by P ≤ 0.05 in the other cohort considered significant.
Results
In one cohort, a polymorphism in DOT1L (rs2269879) was strongly associated with greater systolic (P = 0.0002) and diastolic (P = 0.0016) blood pressure response to hydrochlorothiazide in Caucasians. However, this association was not replicated in the other cohort. When untreated blood pressure levels were analyzed, we found directionally similar associations between a polymorphism in MLLT3 (rs12350051) and greater untreated systolic (P < 0.01 in both cohorts) and diastolic (P < 0.05 in both cohorts) blood pressure levels in both cohorts. However, when further replication was attempted in a third hypertensive cohort and in smaller, normotensive samples, significant associations were not observed.
Conclusions
Our data suggest polymorphisms in DOT1L, MLLT3, SIRT1, and SGK1 are not likely associated with blood pressure response to HCTZ. However, a possibility exists that rs2269879 in DOT1L could be associated with HCTZ response in Caucasians. Additionally, exploratory analyses suggest rs12350051 in MLLT3 may be associated with untreated blood pressure in African-Americans. Replication efforts are needed to verify roles for these polymorphisms in human blood pressure regulation.
doi:10.1186/1479-5876-10-56
PMCID: PMC3320544  PMID: 22440088
Pharmacogenomics; Pharmacogenetics; hydrochlorothiazide; hypertension; blood pressure; DOT1L; SIRT1; MLLT3; SGK1; histone methylation
11.  Genetic Variation in the Beta 2 Subunit of the Voltage-Gated Calcium Channel (CACNB2) and Pharmacogenetic Association with Adverse Cardiovascular Outcomes in the International Verapamil SR-Trandolapril Study-Genetic Substudy (INVEST-GENES) 
Background
Polymorphisms (SNPs) within the regulatory β2 subunit of the voltage-gated calcium channel (CACNB2) may contribute to variable treatment response to antihypertensive drugs and adverse cardiovascular outcomes.
Methods and Results
SNPs in CACNB2 from 60 ethnically diverse individuals were identified and characterized. Three common SNPs (rs2357928, rs7069292 and rs61839258) and a GWAS identified intronic SNP (rs11014166) were genotyped for a clinical association study in 5,598 hypertensive patients with coronary artery disease randomized to a beta-blocker (BB) or a calcium channel blocker (CCB) treatment strategy in INVEST-GENES. Reporter gene assays were conducted on the promoter SNP showing association with clinical outcomes. Twenty-one novel SNPs were identified. A promoter A>G SNP (rs2357928) was found to have significant interaction with treatment strategy for adverse cardiovascular outcomes (p for interaction = 0.002). In Caucasians, rs2357928 GG patients randomized to CCB were more likely to experience adverse outcome than those randomized to BB treatment strategy, with adjusted hazard ratio (CCB vs. BB) of 2.35 (1.19-4.66), p = 0.014. There was no evidence for such treatment difference in AG (1.16, 0.75-1.79, p = 0.69) and AA individuals (0.63, 0.36-1.11, p = 0.11). This finding was consistent in Hispanics and African Americans. CACNB2 rs11014166 showed similar pharmacogenetic effect in Hispanics, but not in Caucasians or African Americans. Reporter assay analysis of rs2357928 showed a significant increase in promoter activity for the G allele compared to the A allele.
Conclusions
These data suggest genetic variation within CACNB2 may influence treatment related outcomes in high risk hypertensive patients.
Clinical Trial Registration Information
Clinical trial identifier: NCT00133692, URL: http://clinicaltrials.gov/ct/gui/show/NCT00133692).
doi:10.1161/CIRCGENETICS.110.957654
PMCID: PMC3060561  PMID: 21156931
Genetic variations; CACNB2; hypertension; cardiovascular outcomes; INVEST-GENES
12.  Lack of association between polymorphisms in STK39, a putative thiazide response gene, and blood pressure response to hydrochlorothiazide 
Pharmacogenetics and genomics  2010;20(8):516-519.
STK39 was previously implicated as a hypertension susceptibility gene and is thought to be involved in control of Na+-Cl− cotransporter (NCC) activity. STK39 has been implicated as a putative thiazide diuretic response gene, as NCC activity is inhibited by thiazides. Thus, we aimed to determine whether STK39 is a thiazide response gene. 195 “good” and 194 “poor” responders to hydrochlorothiazide (HCTZ) were genotyped for approximately 100 single nucleotide polymorphisms (SNPs) within 5000 bases of STK39. SNPs meeting criteria for advancement to replication analysis (P<0.01), along with those previously associated with hypertension, were then analyzed in a second population of 201 HCTZ-treated hypertensives. Two SNPs met these criteria and were analyzed for replication. However, neither these, nor previously implicated SNPs significantly associated with blood pressure response to HCTZ. These data suggest common variants in STK39 likely do not have a clinically relevant role in blood pressure response to HCTZ in hypertensives.
doi:10.1097/FPC.0b013e32833b5958
PMCID: PMC2922977  PMID: 20555294
STK39; SPAK; hypertension; diuretics; hydrochlorothiazide; blood pressure; pharmacogenetics; thiazides; single nucleotide polymorphisms
13.  CACNA1C gene polymorphisms, cardiovascular disease outcomes and treatment response 
Background
The gene encoding the target of calcium channel blockers, the α1c-subunit of the L-type calcium channel (CACNA1C) has not been well characterized and only small pharmacogenetic studies testing this gene have been published to date.
Methods and Results
Resequencing of CACNA1C was performed followed by a nested case-control study of the INternational VErapamil SR/trandolapril STudy (INVEST) GENEtic Substudy (INVEST-GENES). Of 46 polymorphisms identified, eight were assessed in the INVEST-GENES. Rs1051375 was found to have a significant interaction with treatment strategy (p=0.0001). Rs1051375 A/A genotype was associated with a 46% reduction in the primary outcome among those randomized to verapamil SR treatment compared to atenolol treatment (OR 0.54 95% CI 0.32-0.92). In heterozygous A/G individuals, there was no difference in the occurrence of the primary outcome when randomized to verapamil SR versus atenolol treatment (OR 1.47 95% CI 0.86-2.53), while homozygous G/G individuals had a greater than 4-fold increased risk of the primary outcome with verapamil treatment compared to those randomized to atenolol treatment (OR 4.59 95% CI 1.67-12.67). We did not identify allelic expression imbalance or differences in mRNA expression in heart tissue by rs1051375 genotype.
Conclusions
Variation in CACNA1C is associated with treatment response among hypertensive patients with stable coronary artery disease. Our data suggest a genetically-defined group of patients that benefit most from calcium channel blocker therapy, a group that benefits most from β-blocker therapy, and a third group in which calcium channel blocker and β-blocker therapy are equivalent.
doi:10.1161/CIRCGENETICS.109.857839
PMCID: PMC2761685  PMID: 20031608
genetics; pharmacology; ion channels; calcium; pharmacogenetics
14.  ALTERED BETA2 ADRENERGIC RECEPTOR GENE EXPRESSION IN HUMAN CLINICAL HYPERTENSION 
Objectives
The beta 2-adrenergic receptor is involved in mediating vasodilatation via neurohumoral and sympathetic nervous system pathways. Alterations in beta 2-adrenergic receptor gene expression (mRNA transcription) may contribute to the hypertensive phenotype. Human gene expression in clinical phenotypes remains largely unexplored due to ethical constraints involved in obtaining human tissue. We devised a method to obtain normally discarded internal mammary artery tissue from coronary artery bypass graft patients. We then investigated differences in hypertensive and normotensive subjects’ beta 2-adrenergic receptor gene expression in this tissue.
Methods
We collected arterial tissue samples from 46 coronary artery bypass patients in a surgical setting. Using 41 of the samples, we performed TaqMan® RT-PCR and used the delta delta cycle threshold (ΔΔCt) relative quantitation method for determination of fold-differences in gene expression between normotensive and hypertensive subjects. The beta 2-adrenergic receptor target was normalized to glyceraldehyde-phosphate dehydrogenase.
Results
Subjects with hypertension had significantly less expressed beta 2-adrenoceptor gene (2.76-fold, p < .05) compared to normotensive subjects. After Bonferroni correction, gene expression did not differ by race, gender, type/dose of beta-blocker prescribed, positive family history of hypertension, or diagnosis of diabetes mellitus type 2.
Conclusions
These data support the possibility of a molecular basis for impaired adrenoceptor-mediated vascular tone in hypertension. Modification and extension of this research is required.
doi:10.1177/1099800409332538
PMCID: PMC2805083  PMID: 19254913
Beta 2-adrenergic receptor; gene expression; transcription; hypertension; coronary artery
15.  Genotyping the GGGCGG Tandem Repeat Promoter Polymorphism in the 5-Lipoxygenase Enzyme Gene (ALOX5) by Pyrosequencing Assay 
Aims: Efficient genotyping methods for many biologically significant repeat genetic polymorphisms, particularly in GC-rich regions of the genome, are limited. In particular, a short tandem repeat polymorphism [GGCGGG] in the promoter region of ALOX5 has been implicated as an important marker for inflammatory diseases. We developed a pyrosequencing assay to genotype the ALOX5 short tandem repeat polymorphism using pyrosequencing technology that will make assessing this important genetic marker in large, diverse populations more accessible than using current methods. Materials and Methods: We used a nested polymerase chain reaction approach to amplify DNA for pyrosequencing. Population allele frequencies were assessed in two cohorts of previously collected human DNA samples with 188 and 1032 samples, respectively. Sixteen genetic samples with known genotypes were used to confirm the accuracy of the method. Results and Discussion: Genotypes were 100% concordant with samples of known genotype. Genotype frequencies in European American, Hispanic, and African American agreed with previously published results (wild-type homozygotes 66%, 64%, and 19%, respectively). The method presented here will facilitate both genetic association and pharmacogenomic research on this polymorphism in large samples that are ethnically and/or racially admixed.
doi:10.1089/gtmb.2008.0103
PMCID: PMC2998028  PMID: 19473080
16.  Factors Influencing Blood Pressure Response to Trandolapril Add-On Therapy in Patients Taking Verapamil SR (from the International Verapamil SR/Trandolapril [INVEST] Study) 
The American journal of cardiology  2007;99(11):1549-1554.
Factors such as age and race/ethnicity might influence blood pressure (BP) response to drugs. Therapeutic response to the angiotensin-converting enzyme inhibitor trandolapril used as add-on therapy to stable calcium channel blocker therapy with verapamil sustained release 240 mg was addressed in a racially/ethnically diverse group of 1,832 hypertensive patients with coronary artery disease. Furthermore, the association with a polymorphism (1166A→C) in the angiotensin II type 1 receptor gene (AGTR1) was tested. BP response was compared between groups using analysis of covariance after adjustment for covariates associated with BP response. Genotyping was performed using polymerase chain reaction and pyrosequencing. Trandolapril decreased mean unadjusted systolic and diastolic BPs by −9.1 ± 17.3 (SD) and −4.1 ± 10.1 mm Hg, respectively. The percentage of patients with BP under control (<140/90 mm Hg) increased from 6.7% to 41.3% (p <0.0001). Adjusted BP response was significantly associated with age and baseline systolic and diastolic BP (p <0.0001). Whereas the decrease in systolic BP was more pronounced in younger patients, the opposite was observed for diastolic BP decrease. Diastolic BP response was also significantly associated with race. Specifically, the adjusted diastolic BP decrease was significantly smaller in Hispanics and blacks than whites (p = 0.0032 and p = 0.0069, respectively). However, Hispanics achieved a decrease in systolic BP and an increase in BP control similar to the other ethnic groups. There was no genetic association between AGTR1 1166A→C genotype and BP response. In conclusion, trandolapril add-on therapy was effective in increasing BP control, with age and baseline BP associated with both systolic and diastolic BP response. Race was associated with diastolic BP response, although the difference is likely not to be clinically significant and AGTR1 genotype was not associated with BP response.
doi:10.1016/j.amjcard.2007.01.029
PMCID: PMC2720593  PMID: 17531579
17.  α-ADDUCIN POLYMORPHISM ASSOCIATED WITH INCREASED RISK OF ADVERSE CARDIOVASCULAR OUTCOMES: RESULTS FROM INVEST-GENES 
American heart journal  2008;156(2):397-404.
Background
The α-adducin (ADD1) Gly460Trp polymorphism has been associated with hypertension and response to diuretic therapy, but controversy exists.
Methods
The present study was conducted to prospectively investigate the relationship between the ADD1 Gly460Trp polymorphism, diuretic use, and adverse cardiovascular outcomes among 5,979 hypertensive coronary artery disease patients, who participated in the INternational VErapamil SR-trandolapril STudy (INVEST) and provided genomic DNA. The primary outcome was defined as first occurrence of nonfatal stroke, nonfatal myocardial infarction, or all-cause death. Secondary outcomes were the components of the primary outcome. Ancestry informative markers were used to control for population stratification.
Results
In Blacks, ADD1 variant carriers were at higher risk for a primary outcome event than wild-type homozygotes (adjusted hazard ratio (HR) 2.62; 95% confidence interval (CI) 1.23–5.58; p = .012), with a similar trend in Whites and Hispanics, albeit a smaller magnitude of effect (adjusted HR 1.43, 0.86–2.39 in Hispanics; 1.24, 0.90–1.71 in Whites). Secondary outcome analysis showed that the all-cause death was driving the differences in primary outcomes by genotype. There was no interaction between the ADD1 polymorphism and diuretic use for either primary outcome or secondary outcomes.
Conclusions
In hypertensive patients with coronary artery disease, black ADD1 variant carriers showed a 2.6-fold excess risk for a primary outcome event and an 8-fold increase risk of death. White and Hispanic ADD1 variant carriers showed an increased but nonsignificant excess risk. However, the effect of diuretic use on risk of cardiovascular outcomes did not vary by ADD1 carrier status.
doi:10.1016/j.ahj.2008.03.007
PMCID: PMC2547143  PMID: 18657677
pharmacogenetics; ADD1; diuretics; cardiovascular outcomes
18.  Single nucleotide polymorphism discovery and haplotype analysis of Ca2+-dependent K+ channel beta-1 subunit 
Pharmacogenetics and genomics  2007;17(4):267-275.
The large-conductance, Ca2+-dependent K+ channel plays a key role in the control of vascular tone. Variation in the gene encoding the β-1 subunit of the Ca2+-dependent K+ channel (KCNMB1) has been reported to be associated with hypertension, however, variants in KCNMB1 have not been systematically characterized to date. In this study, we have performed the most comprehensive evaluation to date of single nucleotide polymorphisms in KCNMB1 using genomic DNA from 60 individuals of European, African and native American ancestry. We identified and characterized single nucleotide polymorphisms in the exons, intron/exon junctions, upstream region and 3′ untranslated regions of KCNMB1 using denaturing high-performance liquid chromatography combined with direct DNA sequencing. A total of 25 single nucleotide polymorphisms in KCNMB1 were identified. Seven of the polymorphisms (28%) are novel single nucleotide polymorphisms not reported previously. Allele frequencies range from less than 1.7 to 50% and 19 single nucleotide polymorphisms had a minor allele frequency greater than 5%. A lack of strong linkage disequilibrium among the 25 single nucleotide polymorphisms was observed in all three race/ethnicity groups; therefore the identification of haplotype ‘tag’ single nucleotide polymorphisms for genetic association studies is not likely to be appropriate for KCNMB1. Multiple species comparative analysis and in-silico functional analysis were performed to identify potential functionally important single nucleotide polymorphisms within the gene. These data highlight that a tag single nucleotide polymorphism approach will not be appropriate for the study of genes such as KCNMB1, although potentially important functionally significant single nucleotide polymorphisms are suggested for future studies investigating the influence of this gene’s variability on disease and drug response.
doi:10.1097/FPC.0b013e3280105235
PMCID: PMC2713575  PMID: 17496725
Ca2+-dependent K+ channel; haplotype; KCNMB1; single nucleotide polymorphisms
19.  KCNMB1 genotype influences response to verapamil SR and adverse outcomes in the INternational VErapamil SR/Trandolapril STudy (INVEST) 
Pharmacogenetics and genomics  2007;17(9):719-729.
Objectives
We sought to determine whether polymorphisms in the large-conductance calcium and voltage-dependent potassium (BK) channel β1 subunit gene, KCNMB1, are associated with blood pressure response to verapamil SR or adverse outcomes in the GENEtic substudy of the INternational VErapamil SR/trandolapril STudy (INVEST-GENES).
Background
KCNMB1 is involved in calcium sensitivity and hypertension. The association between variability in KCNMB1 and calcium antagonist response, however, has not been assessed.
Methods
Genetic samples were collected from 5979 patients in INVEST. Blood pressure response to verapamil SR and time to achieve blood pressure control was assessed in relation to Glu65Lys and Val110Leu genotypes. The primary outcome (all cause mortality, nonfatal myocardial infarction or nonfatal stroke) was compared between genotype groups, and interaction with verapamil SR therapy was assessed.
Results
Systolic blood pressure response to verapamil SR did not differ by KCNMB1 genotype. Lys65 variant carriers, however, achieved blood pressure control earlier than Glu65Glu individuals [1.47 (interquartile ratio 2.77) versus 2.83 (interquartile ratio 4.17) months, P = 0.01] and were less likely to require multiple drugs at the time of blood pressure control (adjusted odds ratio 0.43, 95% confidence interval 0.19–0.95). Leu110 variant carriers had a reduced risk of primary outcome (hazard ratio 0.68, 95% confidence interval 0.47–0.998). Subgroup analysis revealed this finding to be more pronounced in verapamil SR-assigned patients (hazard ratio 0.587, 95% confidence interval 0.33–1.04) compared with atenolol-assigned patients (hazard ratio 0.946, 95% confidence interval 0.56–1.59). No difference was seen in the occurrence of the primary outcome compared by codon 65 genotype.
Conclusions
Our findings suggest that KCNMB1 genotype influences responsiveness to verapamil SR and risk of adverse cardiovascular outcomes.
doi:10.1097/FPC.0b013e32810f2e3c
PMCID: PMC2713584  PMID: 17700361
KCNMB1; polymorphism; verapamil SR

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