Molecular Biology Reports

, Volume 34, Issue 1, pp 47–52

Angiotensin-converting enzyme gene polymorphism and allele frequencies in the lebanese population: prevalence and review of the literature

Authors

  • Amira S. Sabbagh
    • Department of Pathology and Laboratory MedicineAmerican University of Beirut Medical Center
  • Zaher K. Otrock
    • Department of Internal MedicineAmerican University of Beirut Medical Center
  • Ziyad R. Mahfoud
    • Department of Epidemiology and Population HealthAmerican University of Beirut Medical Center
  • Ghazi S. Zaatari
    • Department of Pathology and Laboratory MedicineAmerican University of Beirut Medical Center
    • Department of Pathology and Laboratory MedicineAmerican University of Beirut Medical Center
Original Paper

DOI: 10.1007/s11033-006-9013-y

Cite this article as:
Sabbagh, A.S., Otrock, Z.K., Mahfoud, Z.R. et al. Mol Biol Rep (2007) 34: 47. doi:10.1007/s11033-006-9013-y

Abstract

We studied the distribution of the D/D, I/D, and I/I genotypes of the angiotensin-converting enzyme (ACE) in a sample of healthy Lebanese individuals to assess their prevalence and compare them with other populations. ACE genotypes were determined using the Cardiovascular Disease (CVD) StripAssay, which is based on a Polymerase Chain Reaction-Reverse hybridization technique. DNA from 133 unrelated healthy donors from our HLA-bank was used. The prevalence of D/D, I/D, and I/I genotypes was found to be 39.1, 45.1, and 15.8% respectively, with D and I allelic frequency of 61.7 and 38.3%, respectively. The sampled Lebanese population showed ACE genotypic distributions similar to Caucasians; however, with tendency towards harboring high D allele frequency together with a low I allele frequency just like the Spanish population. This first report from Lebanon will serve as a baseline statistical data for future investigations of the prevalence of ACE genotypes in association with various clinical entities notably cardiovascular diseases. The medical literature was also reviewed in this context.

Keywords

ACEPrevalenceGenotypingPolymorphismLebanese population

Introduction

During the last two decades there has been an increasing interest in the study of the angiotensin-converting enzyme (ACE) gene and its different polymorphisms. Both in vivo and in vitro studies suggest a role for the Renin-Angiotensin system in hemostasis-regulating mechanisms [1]. Human ACE, present in the liver, kidney, and brain is a dipeptidyl carboxypeptidase that is located on the luminal surfaces of capillary endothelial cells in high concentrations and in the plasma at lower concentrations. ACE cleaves the carboxy-terminal dipeptide of angiotensin I, releasing the physiologically active octapeptide angiotensin II [2]. The latter is a potent vasoconstrictive molecule that plays a key role in modulating vascular tone and is increased locally upon vascular injury [3]. In addition, ACE degrades the vasodilator bradykinin [4], producing a synergistic effect with angiotensin II [5] and a reduction in tissue plasminogen activator production [6].

On the molecular level, the ACE gene is located on the long arm of chromosome 17 (17q23) and is characterized by a polymorphism resulting from the presence or absence of a 287 base pairs fragment of a repeated Alu sequence at intron 16 hence, the corresponding designation of I (for Insertion) or D (for Deletion) of the two resulting alleles [7, 8]. The literature states that as much as 47% of interindividual variability of plasma ACE concentration levels are genetically determined [7, 9, 10]. It is important to state that the level of plasma ACE has an important influence on the pathophysiology of cardiovascular disease and that its different polymorphisms affect the risk of hypertension [11, 12] and other cardiac disease [13, 14].

The three resulting ACE genotypes (D/D, I/D, and I/I) can cause a wide range of ACE activity, with the highest activity being associated with D/D and the lowest with I/I, while the I/D genotype shows intermediate levels. A possible explanation of the higher ACE levels associated with the D allele is that the I allele has a sequence similar to a silencer sequence [7].

To date, the ACE gene polymorphisms have not been studied yet in the Lebanese population. This is the first study that assesses the prevalence of the D and I alleles in addition to D/D, I/D, and I/I genotypes in our community establishing, therefore, a baseline data that may be important for future projects correlating ACE gene polymorphisms and variable clinical conditions.

Materials and methods

Samples and DNA extraction

This study was performed at the American University of Beirut Medical Center (AUBMC), which is a tertiary-care center in Lebanon accommodating patients from the different districts of the country. The Scientific Committee for Research Institutional Review Board at AUBMC approved this study involving the use of archived DNA material. We randomly selected 133 samples from donors logged into our HLA registry and representing healthy Lebanese people originating from different areas and religious communities of the country. Their DNA was originally extracted using the PEL-FREEZ extraction kit (PEL-FREEZ, DYNAL, USA) and stored at −80°C for later use.

PCR and reverse hybridization

To test for the various genotypic profiles of the ACE gene, the CVD StripAssay (ViennaLab, Austria) was used and its protocol was followed as described by the manufacturer. This assay screens for several gene mutations including ACE gene polymorphisms (Fig. 1). Briefly, in vitro, the different gene sequences are simultaneously amplified and biotin-labeled in a single amplification reaction (Multiplexing). The thermocycler program consists of an initial step of 94°C for 2 min, followed by 35 cycles of 94°C for 15 s, 58°C for 30 s, 72°C for 30 s, and a final extension step of 72°C for 3 min. Finally, the amplification products are selectively hybridized to a test strip that contains allele-specific (corresponding to I or D) oligonucleotide probes immobilized as an array of parallel lines. Bound biotinylated sequences are detected using streptavidin-alkaline phosphatase and color substrates.
https://static-content.springer.com/image/art%3A10.1007%2Fs11033-006-9013-y/MediaObjects/11033_2006_9013_Fig1_HTML.gif
Fig. 1

The figure illustrates the orientation of the StripAssay used

Interpretation of results

For each polymorphic position, one of three possible patterns may be obtained: DD, ID, or II genotype.

Results

We studied 133 healthy Lebanese individuals; 70 were males and 63 were females. As listed in Table 1, our data showed that the I/D genotype was the most prevalent (45.1%) followed by D/D (39.1%) and I/I (15.8%). In this Lebanese population sample, the allelic frequencies of each of D and I were calculated to be 61.7 and 38.3%, respectively. Table 2 describes the prevalence of the different ACE genotypes and distribution of D and I alleles as reported in the literature by different investigators. Of interest to note is that the Kuwaiti population holds the lowest frequency ever reported for the I/I genotype (2%) [15].
Table 1

Distribution of ACE genotypes with I and D allele frequencies in 133 unrelated healthy Lebanese individuals

Genotype

N

%

Gender

p-value

Male

Female

D/D

52

39.1

25

27

0.339

I/D

60

45.1

30

30

0.582

I/I

21

15.8

15

6

0.06

Total

133

100

   

Allele

D

164

61.7

80

84

0.443

I

102

38.3

60

42

0.09

Total

266

100

   
Table 2

Frequency of the various ACE genotypes and alleles as reported by selected studies from different population

Study

Population

N

Genotype Frequency (%)

Allele Frequency (%)

DD

ID

II

D

I

Wells et al.

Canadian

300

32.4

47.5

20.1

56

44

Von Depka et al.

German

432

21.3

56.2

22.5

49

51

Hooper at al.

African-American

185

28

55

17

55

45

Uhm et al.

Korean

114

15.8

50

34.2

41

59

Bouba et al.

Greek

102

28.43

50.98

20.59

54

46

Bautista et al.

Colombian

202

26.7

64.4

8.9

59

41

Jackson et al.

British

478

28.2

50.4

21.4

53

47

Fatini et al.

Italian

209

24.4

44.9

30.6

47

53

Gonzalez-Ordonez et al.

Spanish

240

40

44

16

62

38

Rice et al.

White European

279

37.27

43.73

19

59

41

Saeed et al.

Emirati

130

54.6

39.2

6.2

74

26

Al-Eisa et al

Kuwaiti

48

52

46

2

75

25

This study

Lebanese

133

39.1

45.1

15.8

62

38

Discussion

The interaction between the renin-angiotensin system and fibrinolysis has been well established in the medical literature [16] including the prominent effect of the D allele [17, 18]. An interesting pattern of this effect is that it could be exerted according to a sex-specific pattern, for example, the ACE genotype and blood pressure seem to be strongly related in men than in women. Similarly, the D allele was related to venous thrombosis only in male African American subjects [19]. Furthermore, the risk of hypertension is significantly associated with the ACE genotype [20] or the ACE levels [21], but only among males. It is important to note that several studies did not show any association of the ACE genotype neither with hypertension [10, 22, 23] nor with coronary artery disease [24, 25]. In Italian patients, the association between the ACE D/D genotype and coronary artery disease was observed as generally reported for the Caucasians [10, 26, 27] and the Japanese [28]. On the other hand, two large prospective studies on the Danish population [29] and North American male physicians [30] failed to correlate the D allele and the D/D genotype with the incidence of coronary artery disease. Table 2 describes the prevalence of the different ACE genotypes and distribution of D and I alleles as reported in the literature by different investigators.

In a study by Leatham et al., it was found that increased ACE levels might result in a higher risk of acute myocardial infarction among subjects with the D/D genotype [31]. However, in their meta-analysis of 15 studies (including a total of 3394 patients with myocardial infarction and 5479 controls) Samani et al. concluded that the ACE genotype might be associated with myocardial infarction; however, the effect of the D allele was not likely to be high [32]. It is also worth noting that several studies suggested that the impact of the ACE gene polymorphism on the risk of acute myocardial infarction differs from one population to another [33]. In order to explain this trend, some investigators suggested that the effect and impact of the ACE gene polymorphism greatly depend on the genetic make-up of the population under study [34, 35]. Schieffer et al. proposed that there are other existing genetic and environmental factors that could decrease the deleterious effect of the ACE gene in some populations, whereas accentuating it in others [33].

ACE gene polymorphisms and venous thromboembolism (VTE) have also been well studied in the literature with several conflicting results. Some studies showed a high prevalence of the ACE D/D genotype in VTE patients in the absence of hemostasis-related risk factors or in patients with hyperhomocysteinaemia and factor V Leiden mutation [36]. Philipp et al. reported a high prevalence of ACE D/D genotype in patients with thrombosis after orthopedic surgery [37]. The D/D genotype increased the thrombotic risk more than 11-fold while the I/D genotype increased the risk by around 5-fold [37]. On the contrary, Della Valle et al. found that factor V Leiden and ACE I/D polymorphism were not associated with any increased risk of thromboembolic events after total joint arthroplasty in Caucasian subjects [38]. In another study of patients with venous thrombosis after hip replacement surgery, the risk of deep vein thrombosis in those with the D/D genotype was increased more than 10-fold compared with patients with the I/I genotype [37].

Comparing our results with other studies done in the Arab world, we could find two studies from United Arab Emirates [39] and Kuwait [15]. In both studies, the prevalence of the D/D and I/D polymorphisms was comparable to those in our population. However, we had more prevalent I/I polymorphism (Table 2). Our study is the first report from Lebanon that describes the prevalence of ACE gene polymorphisms and the frequency of its alleles. It is recommended that further larger studies be conducted in the Lebanese population based on ACE gene polymorphism and correlating its allele frequencies with the clinical entities notably cardiovascular diseases.

Copyright information

© Springer Science+Business Media, Inc. 2006