Although the angiotensin converting enzyme (ACE) is a strong candidate gene for hypertension, the extensively studied insertion–deletion dimorphism in intron 16 was not found to be associated with it. Several new polymorphisms in the ACE gene were identified, among which a dimorphism in exon 17, ACE G2350A, has a significant effect on plasma ACE concentrations. To assess the value of genotyping the ACE G2350A dimorphism in a genetically homogeneous population, we carried out a retrospective, case–control study of dimorphism G2350A for a putative association with essential hypertension (EH) in a Gulf population (Emirati)–an ethnic group characterized by no alcohol intake and no cigarette smoking. We investigated a sample population of 254 Emirati, comprising 136 normotensive controls, and 118 patients with clinical diagnoses of EH. ACE G2350A alleles were visualized by assays based on polymerase chain reaction and restriction endonuclease analysis. The ACE G2350A dimorphism showed an association with EH (χ2=6.71, 2 df, P=0.05). Further analysis revealed that the ACE G/G 2350 genotype was positively associated (OR=1.06-3.07, P=0.02) with EH. This is the first association study of the ACE G2350A dimorphism with EH, and the positive result might indicate that ACE could be a QTL for EH as originally thought.
Angiotensin-converting enzyme (ACE) is a dipeptidyl carboxypeptidase I (DCP I; 126.96.36.199) that activates angiotensin I through cleavage of the carboxyterminal dipeptide into the potent vasoconstrictor angiotensin II and inactivates the vasodilator peptide bradykinin.1 Both are mediators of vascular tone and smooth muscle cell proliferation.2 Experimental data suggest that the presence of high levels of plasma ACE produce opposing effects mediated by these peptides, which could result in the thickening of the vascular wall eventually leading to the development of vascular disease.1,2 ACE functions as part of the renin–angiotensin system (RAS) for the control of blood pressure (BP). RAS has always been an attractive model system for the study of the genetics of hypertension mainly for its important pathophysiological involvement with BP regulation and the relative ease in directly measuring the intermediate phenotypes. On the basis of the hypothesis that, over the course of a lifetime, even small increases in RAS activity elevate the risk of hypertension in some individuals, ACE gene polymorphisms could be considered a guide to the genetic makeup of hypertension and other cardiovascular diseases (CVD).
Circulating ACE levels show extensive interindividual variability and are highly genetically determined.3,4 An insertion/deletion (I/D) dimorphism, due to the presence or absence of a 287 base pair (bp) alu-type sequence in intron 16 of the ACE gene, has been shown to cosegregate with serum and tissue ACE activities, and that the D allele is associated with elevated ACE levels.3,4,5 Thus, the ACE gene is viewed as a quantitative trait locus (QTL) that modulates circulating ACE levels, and the ACE I/D dimorphism is a marker that is thought to be in linkage disequilibrium (LD) with functional variants located in the ACE gene6,7,8 that are implicated in CVDs.
Several studies on the association of ACE I/D dimorphism and the risk of developing CVDs, such as essential hypertension (EH)9,10,11,12,13,14,15,16,17,18 and myocardial infarction (MI),19,20,21,22,23,24,25,26,27,28 have generated inconsistent information. This instigated a search for new polymorphisms in the ACE gene to identify better markers or actual functional variants. Accumulated evidence points to the existence of two QTLs at this chromosomal locus.29,30,31,32 A genome-scan analysis by The Framingham Heart Study found strong evidence for a QTL on chromosome 17, located close to the ACE gene and linked to BP.16 Among the 13 polymorphisms of the ACE gene recently reported, a dimorphism in exon 17, ACE G2350A, has the most significant effect on plasma ACE concentrations.33 After adjustment for the effect of ACE G2350A dimorphism, the I/D dimorphism was no longer associated with ACE, indicating that it is in LD with ACE G2350A and unlikely to be a functional mutation.33
To assess the value of genotyping of ACE in a genetically homogeneous population, we carried out a retrospective case–control study of dimorphism G2350A for a putative association with EH among nationals from the United Arab Emirates (Emirati)—an ethnic group characterized by no alcohol intake and no cigarette smoking. Our studies aimed at establishing whether the ACE G2350A dimorphism is a genetic marker and an independent risk factor for EH.
Materials and methods
The United Arab Emirates (UAE) is a Federation of seven Emirates (the Abu Dhabi Emirate being the largest) with an indigenous population comprising UAE nationals, who are Gulf Arabs of Bedouin descent. In this pilot, retrospective case–control study, we investigated a sample population of 254 UAE nationals (Emirati) from the Abu Dhabi Emirate recruited from the outpatient clinics of Mafraq Hospital, with a view to identify putative associations between dimorphism G2350A of ACE and EH. This project was approved by the Research Ethics Committee of the Faculty of Medicine and Health Sciences (UAE University, Al Ain, UAE).
Patients were classified as having EH if their systolic blood pressures (SBP) had been above 140 mmHg and their diastolic blood pressures (DBP) above 90 mmHg on at least three separate occasions, and if they had no clinical signs, symptoms and laboratory findings suggestive of secondary hypertension. Controls were selected by matching them for age, gender and BMI (Table 1).
Blood was collected in 10 ml Na–EDTA tubes and kept frozen at −20°C. DNA was extracted using standard protocols34 and stored in 10 mM Tris-HCl, 1 mM EDTA, pH 8.0.
Codon 2350 ACE genotypes were first visualized by a method based on allele-specific oligonucleotides.32 It was then described as ACE-8 by Zhu et al,33 as a modification by introducing a mismatch guanine at the 3′ end of the primer sequence, resulting in the amplification of a BstU1 restriction site. This more convenient method involves a set of primers designed to amplify a 122-bp fragment encompassing the polymorphic region of ACE gene: 5′IndexTerm-CTGACGAATGTGATGGCCGC 3′ (upstream) and 5′IndexTerm-TTGATGAGTTCCACGTATTTCG-3′ (downstream). The PCR contained 100–200 ng DNA template, 125 μM dNTPs, 2.5 mM MgCl2, and 0.3 mM of each primer and 1 U Taq polymerase, in a final volume of 10 μl. After initial denaturation at 95°C for 5 min, PCR was carried out for 35 cycles, each one comprised of denaturation at 94°C for 30 s, annealing at 58°C for 30 s, and extension at 72°C for 30 s, with a final extension time of 10 min at 72°C. The PCR products (5 μl) were digested with 5 U of BstU1 (Life Technologies, Beverly, MA, USA) at 60°C for 2 h. Digested fragments were separated by electrophoresis on 3% agarose gel and identified by ethidium bromide staining. Allele G2350 was visualized as a 122-bp fragment and allele A2350 as 100- and 22-bp fragments (Figure 1).
Statistical analyses were carried out using the SPSS® (Statistical Package for Social Sciences) Software Version 10.0 for Windows® (Gorinchem, The Netherlands). Distribution differences of G/A 2350 genotypes in the patients (EH) as compared to distribution in the control group were assessed by χ2 analyses on 3 × 2 tables. Estimations of departures from the Hardy–Weinberg equilibria (DA) and the tests for statistical significance were calculated as reported by Haviland et al.35 Allele frequencies were compared between the patients and control group by using the two-sided Fisher's exact test. For all analyses, statistical significance was considered when significance level (P) values were lower than 0.05.
This study included 254 UAE national (Emirati) unrelated subjects (52% males) with an overall mean age of 56±12.4 years. The EH patients and controls were age, BMI and gender-matched.
Table 2 shows the data pertaining to both genotype and allele distributions in the two groups of subjects. ACE genotypes did not occur in Hardy–Weinberg proportions neither in the normotensive control group (DA=0.073, χ2=11.92, 1 degree of freedom (df), P<0.001) nor in the hypertensive sample population (DA=0.051, χ2=8.29, df=1, P<0.01). Such deviations are expected in the case of marked association with the clinical phenotype especially in homogenous populations.
Differences in the distributions of the three genotypes according to clinical phenotype were statistically significant (EH vs controls: χ2=6.71, 2 df, P=0.05). Patients having the GG genotype were predisposed to EH (odds ratio of 1.80 with a 95% confidence interval of 1.06–3.07 and P=0.02).
Frequency of the G alleles were 0.74±0.04 in the EH patient group compared to 0.63±0.04 in the normotensive subjects. The 11% difference in the frequency of the G alleles in the two subsets of the Emirati population was statistically significant (F-statistic= 477.81; P<0.001) and indicates a major gene effect in a complex clinical phenotype such as EH.
We also sought to explore the putative associations between the three genotypes (GG, GA, AA), and SBP and DBP. The SBP and DBP values were also not statistically different between the genotypes (Table 3).
Exploring putative associations in various ethnic groups, which may be more genetically homogeneous due to high degrees of consanguinity, minimizes the influence of selection bias and population stratification. The Emirati population that was the subject of this investigation, offered another advantage—absence of alcohol intake and of cigarette smoking, which are the usual confounding, environmental factors in these types of studies.
‘Control’ individuals who were included in this investigation constituted a ‘comparison’ rather than a ‘control’ group. They were indeed free of disease, age-matched with the patients, and chosen because their BMIs matched those of the patients. As these control individuals had neither personal nor family history of EH, they represented a valid comparison group, although it could not be predicted whether some of them would develop EH in future.
Several studies have suggested that the D allele of ACE I/D dimorphism confers increased risk of developing CVDs.7,22,25 At the same time, considerable negative evidence exists on this question. An analysis of 11 000 cases and controls showed no relationship between MI and the I/D dimorphism.26 The I/D marker was likewise not associated with MI in Italian and French sample populations.8,19 O'Malley et al20 summarized the association between the I/D dimorphism and CVD risk, grouping studies by geographical region and disease prevalence. In this analysis, the ACE I/D dimorphism did not appear to be a clinically useful indicator of risk for MI. Although hypertension is an important risk factor for CVD, conflicting results have been reported regarding the association between the I/D dimorphism and BP9,10,12,14,17 as has been the case for MI. Two large population-based studies found only marginally significant evidence of association and linkage, for BP and the I/D dimorphism.12,16
Plasma ACE levels are remarkably stable within an individual, while marked differences are observed between individuals.3,4 The results of a combined segregation-linkage analysis in French families suggested that the I/D dimorphism was in strong LD with an unmeasured functional mutation of the ACE gene.6 This mutation appeared to be frequent and explained the major part of the genetic variance of plasma ACE. A similar study performed in African Caribbean families confirmed the existence of an ACE-linked QTL influencing ACE levels, but also revealed a weaker LD between this QTL and the I/D dimorphism and suggested the existence of a second QTL unlinked to ACE.31,32 Zhu et al33 reported 13 polymorphisms in the ACE gene using linkage and association studies. The polymorphism in exon 17, ACE G2350A, had the most significant effect on plasma ACE concentration, accounting for 19% of the total variance in ACE plasma levels. After adjustment for the effect of ACE G2350A dimorphism, the I/D polymorphism was not found to be associated with plasma ACE concentration, indicating that it is in LD with ACE G2350A and unlikely to be a functional mutation. Besides the effect on plasma ACE concentration, Zhu et al33 reported, that this dimorphism was significantly associated with SBP with an average increase of 3.2 mmHg with each copy of the G allele. Although this effect was not observed in our study, subjects having the GG genotype had on average approximately 10 mmHg higher SBP values compared to those having GA or AA genotypes. Large standard deviations could have resulted in the trend not being statistically significant.
We report the allele frequency of the ACE G2350A dimorphism in the Emirati population to be 0.63±0.04 and 0.37±0.04 for the G2350 and A2350 alleles, respectively. In the present study, G2350A genotype distributions did not occur in Hardy–Weinberg proportions in the control group or in the EH patient group as indicated by the DA statistics. This observation assumes increased significance in view of the fact that the Emirati population is very homogenous due to high consanguinity. The Hardy–Weinberg disequilibrium is observed especially in homogenous populations when there is strong cosegregation of the marker with the clinical phenotype. The cosegregation of the marker with the disease induces a disequilibrium in the control population as well. This is yet another indication that ACE is likely to be a major QTL for EH.
Our data on the Emirati population show a significant association between G2350A dimorphism and EH. The GG genotype was significantly associated with increased risk for EH. This observation indicates that ACE G2350A dimorphism may be the functional mutation of the ACE gene responsible for EH through elevated plasma ACE levels.
If confirmed, these results would support the ‘RAS hypothesis’ of hypertension predisposition and would help to reconcile previous, inconsistent findings. In spite of its association with EH, the ACE G2350A dimorphism may very well not be the actual functional mutation. Instead, it could be located near another functional mutation, as yet undiscovered, in ACE or any other gene that could be in LD with it in this region of chromosome 17.
In summary, despite these caveats, our data support that the G2350A dimorphism is associated with EH and can be used as an independent risk marker in the Emirati population. This is the first association study of the ACE G2350A dimorphism with EH and provides an indication that the ACE gene could be a QTL underlying individuals' genetic susceptibility to EH. Of course, several other genes are likely to participate in BP dysregulation and EH phenotypes. Given the complex nature of genetic susceptibility for chronic degenerative diseases, further studies need to be conducted in individual ethnic groups to verify the disease relevance of this polymorphism.
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We thank Dr Anwar Ali Siddiqui for kindly reviewing the manuscript and providing us with immense support at the Juma Research Lab, which made this work possible.
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Saeed Mahmood, M., Saboohi, K., Osman Ali, S. et al. Association of the angiotensin-converting enzyme (ACE) gene G2350A dimorphism with essential hypertension. J Hum Hypertens 17, 719–723 (2003). https://doi.org/10.1038/sj.jhh.1001600
- angiotensin converting enzyme
- polymerase chain reaction
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