Promoter polymorphisms in ACE (angiotensin I-converting enzyme) associated with clinical outcomes in hypertension

Abstract

Genetic variants of ACE are suspected risk factors in cardiovascular disease, but the alleles responsible for the variations remain unidentified. To search for regulatory polymorphisms, allelic angiotensin I-converting enzyme (ACE) mRNA expression was measured in 65 heart tissues, followed by genotype scanning of the ACE locus. Marked allelic expression imbalance (AEI) detected in five African-American subjects was associated with single-nucleotide polymorphisms (SNPs) (rs7213516, rs7214530, and rs4290) residing in conserved regions 2-3 kb upstream of ACE. Moreover, each of the SNPs affected transcription in reporter gene assays. SNPs rs4290 and rs7213516 were tested for associations with adverse cardiovascular outcomes in hypertensive patients with coronary disease (International Verapamil SR Trandolapril Study Genetic Substudy (INVEST-GENES), n = 1,032). Both SNPs were associated with adverse cardiovascular outcomes, largely attributable to nonfatal myocardial infarction in African Americans, showing an odds ratio of 6.16 (2.43-15.60) (P < 0.0001) for rs7213516. The high allele frequency in African Americans (16%) compared to Hispanics (4%) and Caucasians (<1%) suggests that these alleles contribute to variation between populations in cardiovascular risk and treatment outcomes.

Figures

Figure 1

ACE gene structure (University of California–Santa Cruz genome browser) and location of polymorphisms tested in this study. Overviews of HapMap linkage disequilibrium (LD) in the gene region for individuals from Utah of northern-European ancestry (CEU) and individuals from Yoruba, Nigeria (YRI), are shown at bottom (Haploview). ACE, angiotensin I–converting enzyme.

Figure 2

ACE allelic mRNA expression in left ventricular heart tissues from (a) African Americans and (b) Caucasians. Allelic mRNA expression ratios (major/minor allele for marker single-nucleotide polymorphisms (SNPs) rs4309 (C/T), rs4343 (A/G)) are averages of results using both markers. Allelic expression imbalance was prevalent in (a) African-American but not (b) Caucasian heart tissues. Genotypes for the promoter SNPs are indicated above the African-American samples. Data are mean ± SD, ***P < 0.001 vs. pooled DNA ratios. ACE, angiotensin I–converting enzyme.

Figure 3

Total mRNA expression levels of angiotensin I–converting enzyme (ACE) in 65 heart tissues. Box plots display median expression ± one quartile. Results are grouped by (a) genotype of the insertion/deletion (I/D) (P = 0.93) and (b) carriers of the promoter rs4290 T allele. ACE mRNA levels are relative to β-actin. *P < 0.05 vs. CC genotype.

Figure 4

Luciferase reporter gene assay of the ACE promoter in bovine aortic endothelial cells (BAEC) (a) and HEK293 cells (b). An ACE promoter fragment from −4,335 to +1 was cloned into pGL3.basic vector containing the promoter single-nucleotide polymorphisms (SNPs) rs7213516 (G/A), rs7214530 (T/G), and rs4290 (C/T). The reference haplotype was G-T-C, whereas the variant constructs contained one to three minor alleles. In BAEC, 0.8 μg plasmids were cotransfected with 40 ng Renilla luciferase plasmid using either Lipofectamine or FuGENE reagent. Luciferase activities from fused-pGL3 vector were normalized using Renilla luciferase activity as an internal control. *P < 0.05; **P < 0.001 compared to reference haplotype G-T-C. In HEK293 cells, various amounts of plasmid were transfected using Lipofectamine, with no differences observed between any conditions. ACE, angiotensin I–converting enzyme.

Figure 5

Linkage disequilibrium (LD) structure of polymorphisms in the International Verapamil SR Trandolapril Study Genetic Substudy clinical genetic association study. Values for D′ and r2 are shaded on a gradient based on the strength of correlation. Boxes without number values and shaded dark gray indicate perfect correlation. Boxes without number values and shaded light gray indicate that low allele frequency prevented calculation of D′.

Figure 6

Odds ratios of three polymorphisms for primary outcome in the overall population and within each group. Odds ratios were adjusted for age, sex, race/ethnicity, body mass index, smoking, International Verapamil SR Trandolapril Study treatment strategy, previous myocardial infarction, previous stroke, heart failure, diabetes, renal insufficiency, baseline systolic blood pressure, diuretic use, and angiotensin I–converting enzyme inhibitor use. AA, African American; Cauc, Caucasian; Hisp, Hispanic.

Figure 7

Promoter sequence alignments and transcription factor–binding sites. The three promoter single-nucleotide polymorphisms rs7213516, rs7214530, and rs4290 are located −2883, −2828, and −2306 bp upstream of the transcription start site (+1). Predicted MEF2A-binding sites (JASPAR position–weight matrices) are shown. Sequence alignments (made with CLUSTALW) are based on Basic Local Alignment Search Tool of the human promoter region. *1-bp insert in rhesus, dog, elephant, and armadillo; †9-bp insert in dog and armadillo.