Association Between Angiotensin-Converting Enzyme Insertion/Deletion Genetic Polymorphism and Hypertension in a Sample of Lebanese Patients

Abstract

Background/Aim:

Several studies have looked at the potential link between angiotensin-converting enzyme (ACE) insertion/deletion (I/D) polymorphism and the risk of hypertension and have shown that the DD polymorphism may be associated with a higher prevalence of hypertension. Our objective was to assess for possible association between ACE variants and hypertension in a sample of Lebanese patients.

Methods:

One hundred ninety-two Lebanese subjects were included. DNA was isolated and amplified by polymerase chain reaction. The products were identified by gel electrophoresis according to their size.

Results:

One hundred fifteen (59.9%) patients were hypertensive and 77 (40.1%) were nonhypertensive with the following genotype frequencies: 43.4% DD, 45.2% ID, and 11.4% II compared with 35.2% DD, 51.9% ID, and 12.9% II, respectively. Age was found to be the most significant risk factor for hypertension. This was more prominent when accounting for ACE genotype; for instance, the DD genotype with age had a significantly higher odds ratio (OR = 11.852; p = 0.001) than the ID genotype with age (OR = 4.599; p = 0.006), II genotype with age (OR = 1.866; p = 0.519), and age alone (OR = 5.558; p = 0.006).

Conclusion:

Our results show that the ACE I/D polymorphism is common in Lebanon, and the combinations of ACE D allele and age is associated with an increased risk of hypertension.

Introduction

Hypertension is a common chronic medical condition characterized by an elevated systolic and/or diastolic blood pressure (Boulpaep, 2003). It affects about 20% of the adult population and it has been estimated that 1.56 billion patients will suffer from hypertension in the year 2025 (Adrogue and Madias, 2007). Hypertension is also quite prevalent in developing countries such as Lebanon; for instance, it was shown that 23.1% of the Lebanese population suffers from hypertension (Tohme et al., 2005).

There are two types of hypertension: secondary hypertension, the least prevalent, is due to several pathophysiological conditions such as hyperthyroidism, kidney disease, and adrenal disease, whereas essential (primary) hypertension, the most prevalent, is a multifactorial disease that results from the interaction between a multitude of environmental and genetic factors (Kotchen, 2009). The risk of developing essential hypertension increases with advancing age; for instance, almost 50% of subjects between 60 and 69 years of age are diagnosed with hypertension, and the risk increases further in patients who are 70 years of age and older (Hoffman, 2007). Irrespective of the underlying mechanism, persistent elevation in systemic blood pressure is a major risk factor for cardiovascular diseases such as coronary artery disease, heart failure, and cerebrovascular disease (Hoffman, 2007).

Angiotensin-converting enzyme (ACE) is a dipeptidyl carboxy peptidase belonging to the class of zinc metalloproteases. It is found as a membrane-bound protein in endothelial, epithelial, and neuroepithelial cells in the liver, kidney, and brain. It is involved in the cleavage of many molecules, the most important of which is the physiologically inactive angiotensin I molecule, which is converted to the active angiotensin II molecule. This protein plays a crucial role in high blood pressure development (Kotchen, 2009).

In humans, the ACE gene is encoded by a 21-kb area located on chromosome 17 (17q23). This gene comprises 26 exons and 25 introns (Hubert et al., 1991). A common variant of this gene is characterized by an insertion/deletion (I/D) polymorphism (rs1799752-rs4340-rs13447447-rs4646994) emanating from the presence or absence of a 287-base-pair fragment of a repeated Alu sequence on intron 16 resulting in 3 genotypes: II, ID, and DD (Hubert et al., 1991). It has been shown that the D allele is associated with an increased ACE activity and hence a potential increase in the risk of hypertension (Rigat et al., 1990).

Several studies investigated the potential association between the ACE DD genotype and hypertension and showed inconsistent results across different populations and ethnicities. A significant association between ACE DD genetic polymorphism and hypertension was reported in American Caucasians and patients from East Asia such as Indians and Chinese (Mastana and Nunn, 1997; Abbud et al., 1998; Das et al., 2008). However, this association was not found in European populations (Frossard et al., 1997; Zaman et al., 2001; Companioni et al., 2007).

In essence, although there is evidence of correlation between ACE genetic I/D polymorphism and catalytic activity of the enzyme, there are obvious ethnic differences in association with hypertension development. Therefore, it appears that one must be quite careful in extrapolating results from a specific ethnic group of subjects to another. One interesting and understudied group is the Arab population in general and the Lebanese people in particular.

To date, few ACE genetic I/D polymorphism frequency studies have been performed on Arabs (Al-Eisa et al., 2000; Bayoumi et al., 2006; Al-Gasham et al., 2009; Salem and Batzer, 2009), and to our knowledge, the association between ACE variants and hypertension was evaluated in the Emirates only (Frossard et al., 1997). As for Lebanon, Sabbagh et al. (2007) determined the frequency of ACE polymorphism in 133 healthy Lebanese subjects and showed that the D variant is common with an allelic frequency of 62%. Therefore, the present study was undertaken to explore a potential association between ACE DD genetic polymorphism and hypertension in a sample of Lebanese patients.

Materials and Methods

Study population

This study included 192 Lebanese patients who were initially recruited at the American University of Beirut Medical Center for the evaluation of the pharmacogenetics of anticoagulants. A questionnaire was filled out, and retrospective clinic and hospital chart reviews were performed. Baseline demographics, occupational exposure, life style habits, and medical history were collected. Patients were considered to have essential hypertension based on the physicians and nurses notes and/or evidence of use of antihypertensive drugs obtained from clinic and hospital medical records. Patients whose medical charts did not state “hypertension” as a diagnosis and were not taking antihypertensive medications were considered normotensive. The study was approved by the University Institutional Review Board for human rights.

Genotyping

Venous blood was drawn in EDTA-containing tubes and stored at −80°C until analysis. DNA was isolated using a DNA isolation kit from Qiagen (Germantown, MD) according to the manufacturer's guidelines but with minor changes. Genomic DNA was amplified using polymerase chain reaction (PCR). PCR was started with an initial denaturation step at 95°C for 2 min. The DNA was then amplified for 30 cycles with denaturation at 94°C for 30 s, annealing at 59°C for 30 s, and extension at 72°C for 45 s. This was followed by a final extension at 72°C for 9 min.

The PCR products were visualized on a 2% agarose gel. The method and primer sequences were based on the publication of Rigat et al. (1990). Homozygosity for the D allele (DD) was identified by the presence of a single 190-bp PCR product, homozygosity for the I allele (II) was identified by the presence of a single 490-bp PCR product, and heterozygosity (ID) was identified by the presence of both 190- and 490-bp PCR products.

Statistical analysis

Data were analyzed using the statistical package SPSS v.16 (SPSS, Chicago, IL). Means, standard deviations, and frequencies were calculated for the baseline characteristics of the subjects (Table 1). ACE genotypes and allele frequencies were computed and tested for Hardy-Weinberg equilibrium. Assessment was made of the relationship between hypertension and patients' characteristics including age, sex, smoking, alcohol intake, dyslipidemia, diabetes mellitus type 2, coronary artery disease (e.g., angina and myocardial infarction), cerebrovascular disease (e.g., stroke and transient ischemic attacks), atrial fibrillation, and ACE genotype using the chi-square test or t-test when applicable (Table 1). Further, a general logistic regression model was used to predict the effect of genetic factors (ACE genotype: input 1 for II, 2 for ID, and 3 for DD) and the nongenetic factors listed in Table 1 on hypertension being the dependent variable. The model was run twice: once with age as a continuous variable and once with age categorized as follows: 1 for age <51, 2 for age ≥51 and ≤80, and 3 for age >81. Another general logistic regression was performed in the whole population sample to assess for only the variables that were shown to be significantly associated with hypertension and then within the three different ACE genotypes. In this analysis, ACE genotype was categorized in three different ways: (1) input 1 for II, 2 for ID, and 3 for DD; (2) input 1 for II and ID combined and 2 for DD; and (3) input 1 for DD and ID combined and 2 for II. Odds ratios (ORs) were generated and were considered statistically significant when the 95% confidence interval (CI) did not include 1. A p-value of <0.05 was considered statistically significant.

Table 1

Clinical Features of Study Participants and ACE Genotype Profile

	All	Nonhypertensive	Hypertensive
	n = 192	n = 77	n = 115	p-Value^a
Age in years (mean ± SD)	67 ± 14	60 ± 15	71 ± 10	0.000
Sex
Males	55.7	76.6	67.7
Females	44.3	23.4	32.3	0.481
Smoking	22.4	10.9	11.5	0.876
Alcohol	24.5	9.9	14.6	0.935
Dyslipidemia	42.2	12	30.2	0.077
Diabetes mellitus	22.4	5.2	17.2	0.069
Coronary artery disease	38.0	10.9	27.1	0.402
Cerebrovascular disease	17.7	5.7	12	0.852
Atrial fibrillation	54.7	15.6	39.1	0.042
ACE genotype
DD	-	35.2	43.4
ID	-	51.9	45.2
II	-	12.9	11.4	0.507

All values are percentages (%) except for age.

p-Values were generated by t-test for age and chi-square for the rest.

Results

This study included a total of 192 Lebanese individuals of whom 115 (59.9%) were hypertensive and 77 (40.1%) were nonhypertensive with the following genotype frequencies: 43.41% DD, 45.24% ID, and 11.35% II compared with 35.16% DD, 51.87% ID, and 12.97% II, respectively (Tables 1 and 2). The frequencies of the ACE alleles were found to fit Hardy-Weinberg Equilibrium.

Table 2.

ACE D Allelic Frequencies in Nonhypertensive Populations

Study	Population	n	D frequency
Bayoumi et al. (2006)	Sudanies	121	0.64
	Emiraties	111	0.73
	Somalies	53	0.61
	Omanies	124	0.71
Salem and Batzer (2009)	Egyptians	164	0.67
	Jordanians	60	0.66
	Syrians	70	0.60
Al-Gasham et al. (2009)	Saudis	273	0.72
Al-Eisa et al. (2001)	Kuwaiti	48	0.75
Gunes et al. (2004)	Turkish	246	0.52
Bouba et al. (2003)	Greek	102	0.54
Von Depka et al. (2003)	German	432	0.49
Jackson et al. (2000)	British	478	0.53
Fatini et al. (2003)	Italian	209	0.47
Rice et al. (1999)	White Europeans	279	0.59
Gonzalez-Ordonez et al. (2000)	Spanish	240	0.62
Sabbagh et al. (2007)	Lebanese	133	0.62
This study	Lebanese	77	0.61

Table 1 shows that aging and being diagnosed with atrial fibrillation were significantly associated with hypertension (p = 0.000 and 0.042, respectively). No significant associations were found between evidence of hypertension and ACE genotype as well as the following nongenetic factors: sex, smoking, alcohol intake, dyslipidemia, diabetes mellitus type 2, coronary artery disease, and cerebrovascular disease. Further, the general regression analysis including all variables listed in Table 1 showed that, again, ACE genotype was not associated with hypertension: OR = 1.156, p = 0.585, CI = [0.687-1.944] and OR = 1.176, p = 0.530, CI = [0.708-1.953] when age was used as a continuous variable or a categorized variable, respectively. Genotype, when used alone in the model, was also not associated with hypertension: OR = 1.256; p = 0.244, CI = [0.814-1.940] (Table 3). Age was found to be significantly associated with hypertension: OR = 1.054, p = 0.001, CI = [1.021-1.089] and OR = 3.260, p = 0.006, CI = [1.411-7.536] when used as a continuous variable or a categorized variable, respectively. Finally, atrial fibrillation was associated with hypertension within the model that used age as a categorized variable only: OR = 2.064, p = 0.042, CI = [1.028-4.145]. There was no association between ACE genotype and incidence of atrial fibrillation (p = 0.167).

Table 3.

Association Between ACE D Genetic Polymorphism and Hypertension by Study

		Nonhypertensive		Hypertensive
Study	Population	n	D frequency	n	D frequency	OR	95% CI	p-Value
Staessen et al. (1997)	Meta-analysis	43036	N/A	6923	N/A	1.1	0.95-1.27	0.19
Stanković et al. (2002)	Serbians	210	0.56	175	0.66	1.58	N/A	0.034
Gunes et al. (2004)	Turkish	63	0.519	183	0.517	N/A	N/A	>0.05^a
Abbud et al. (1998)	Americans	100	0.53	209	0.62	1.45	0.82-2.54	0.035^b
Ismail et al. (2004)	Pakistanis	108	0.55	211	0.55	N/A	N/A	0.413^a
Frossard et al. (1997)	Emiraties	74	0.67	85	0.64	N/A	N/A	0.75^a
Morshed et al. (2002)	Bangladeshi	59	0.457	44	0.693	N/A	N/A	<0.001^a
Das et al. (2008)	Indians	35	0.34	35	0.6	N/A	N/A	<0.001^c
Chiang et al. (1996)	Chinese	115	×^d	157	×^d	1.46	N/A	0.03
Higaki et al. (2000)	Japanese	3814	0.35	1200	0.38	1.15	1.05-1.27	<0.05^e
Gesang et al. (2002)	Tibetans	123	0.36	103	0.49	1.667	1.145-2.428	0.008
This study	Lebanese	77	0.61	115	0.66	1.256	0.814-1.940	0.244^f

Chi-square test.

OR = 1.57 with 95% CI = [1.06 to 2.36] and p = 0.02 after adjusting for age.

OR = 7.483 with 95% CI = [1.746 to 30.192] and p < 0.05 after adjusting for age.

We were unable to get the full article, hence only the abstract that did not include allelic frequencies was reviewed.

OR = 1.75 with 95% CI = [1.21 to 2.53] and p < 0.05 after adjusting for age.

OR for association between age and hypertension is significant and highest within the DD genotype.

N/A, not applicable.

Table 4 shows the ORs for the association between age and hypertension in the whole population sample and within each ACE genotype category. It is apparent that the statistically significant relationship is highest in the DD genotype category and is lost within the II genotype. This is more obvious when the ID and DD genotypes were lumped into 1 category. When assessing for ID and II genotype combinations vs. DD alone, the resultant ORs were statistically significant for both categories, hence asserting that the effect is driven by the D allele (OR = 1.049, p = 0.004, CI = [1.015-1.084] for ID and II and OR = 1.121, p = 0.000, CI = [1.061-1.184] for DD alone when age was used as a continuous variable, and OR = 3.670, p = 0.005, CI = [1.480-9.103] for ID and II and OR = 11.852, p = 0.001, CI = [2.683-52.362] for DD alone when age was used as a categorized variable).

Table 4.

Regression Modeling Association Between Hypertension and Age in the Whole Population Sample and Within ACE Genotype Categories

				95% CI
		OR	p-Value	Lower limit	Upper limit
	All sample	1.074	0.000	1.045	1.104
	II	1.013	0.673	0.956	1.073
	ID	1.065	0.002	1.023	1.109
Age continuous	DD	1.121	0.000	1.061	1.184
	II	1.013	0.673	0.956	1.073
	DD and ID	1.089	0.000	1.054	1.124
	All sample	5.558	0.000	2.591	11.924
	II	1.866	0.519	0.281	12.412
	ID	4.599	0.006	1.558	13.576
Age categorized^a	DD	11.852	0.001	2.683	52.362
	II	1.866	0.519	0.281	12.412
	DD and ID	3.098	0.001	1.632	5.878

Age was categorized as follows: 1 for age <51, 2 for age ≥51 and ≤80, and 3 for age >81.

OR, odds ratio; 95% CI, 95% confidence interval.

Discussion

This is the first study investigating the association of ACE I/D genetic polymorphism with hypertension in a sample of Lebanese patients.

Frequency distribution

In the population under study, the D allele was common in nonhypertensive patients and its frequency was similar to previously reported results (Sabbagh, et al., 2007). It is interesting that the allele frequencies are also comparable to representative countries of the relevant known migration routes to Lebanon (Table 2) (Zalloua et al., 2008; El-Sibai et al., 2009).

Association with hypertension

In the present sample of Lebanese patients, ACE I/D polymorphism was not associated with hypertension; nevertheless, age was found to be the most significant risk factor for hypertension and this was even more prominent when accounting for the D allele of the ACE genotype.

Table 3 shows the results of association studies of ACE I/D polymorphism with hypertension. It is apparent that the results were not similar across different populations; for instance, there was a lack of association between ACE I/D polymorphism and hypertension in the American continent (United States, Cuba) (Abbud et al., 1998; Companioni et al., 2007), most of the European countries (Russia, Deutschland, Greece, Turkey) (Schmidt et al., 1993; Popov et al., 1996; Vassilikioti et al., 1996; Stanković et al., 2002; Gunes et al., 2004), and the Middle East (United Arabic Emirates) (Frossard et al., 1997). However, the D allele was strongly associated with hypertension in the far eastern countries such as Japan, India, China, Tibet, and Bangladesh (Chiang et al., 1996; Higaki et al., 2000; Gesang et al., 2002; Morshed et al., 2002; Das et al., 2008).

It was not a surprise that age was the most important factor associated with hypertension. For instance, it is well known that environmental factors that surround the organism during its development influence the expression of the genetic information at a later stage during the human lifetime (Kunes and Zicha, 2009). This is why several investigators have found significant results after adjusting for age. Thus, Das et al. (2008) computed an OR of 7.483 [1.746-30.192] after adjusting for age in an Indian population. Similarly, Abbud et al. (1998) obtained significant results on American populations when accounting for the age factor. Higaki et al. (2000) from Japan also compared the increased risk of developing hypertension in different age categories. They obtained a higher OR for the DD genotype in elderly subjects (OR = 1.785, [0.848-3.759]) than in younger subjects (OR = 1.585, [0.881-2.849]) (Table 4).

In the present study, atrial fibrillation was found to be associated with hypertension, but this was significant within the logistic regression model that used age as a categorized variable only. It is unlikely that there might be a causal relationship. Atrial fibrillation is not a known risk factor for development of hypertension, yet a patient may be diagnosed with atrial fibrillation and hypertension together, especially at an older age (Marchlinski, 2009). The present sample of Lebanese patients is a convenient sample from a study that initially recruited patients at the American University of Beirut Medical Center for evaluation of the pharmacogenetics of anticoagulants. Therefore, it is possible that the significant relationship found in this study is due to the fact that many of the patients had atrial fibrillation as a reason for anticoagulation and most of them were >50 years of age. Although it has been previously reported that the I/D ACE gene polymorphism may be associated with an increased risk of atrial fibrillation, it was not the case in the present study (Gensini et al. 2003; Ravn et al., 2008).

In contrast to our results, few studies have shown that hypertension is more prevalent in men when compared with women, and hence, investigators have adjusted for sex in their analysis. This is the case for, for example, Higaki et al. (2000), who showed a higher OR of the DD genotype in young men (OR = 1.99, [1.07-3.63]) than in young women (OR = 1.42, [0.72-2.68]).

Similarly, Serbian men were more prone to develop hypertension (OR = 2.05, [1.07-3.91]) than Serbian women (OR = 0.72, [0.33-1.60]) (Stanković et al., 2002); however, these results are not always consistent across different groups, for example, Tibetan women were at a higher risk for developing hypertension (OR = 2.161, [1.313-3.555]) when compared with Tibetan men (OR = 1.177, [0.660-2.098]) (Gesang et al., 2002).

It has been previously reported that ACE polymorphism may be associated with hypercoagulability and hence a potentially increased risk of deep vein thrombosis, especially in patients with thrombophilias (Philipp et al., 1998; La Valle et al., 2001; Fatini et al., 2003). We did not assess for these factors because only 18 of the subjects had thrombophilias of different types and most of them were <50 years of age. Further, only 16 individuals had isolated deep vein thrombosis events and all the rest occurred with other pathologies such as coronary artery disease, atrial fibrillation, and other diseases.

In the present study, only ACE I/D polymorphism was assessed. It is recommended to investigate other potential markers that may be involved in the development of essential hypertension, such as α-adducin, AT1, and adrenergic receptor genes (Bianchi et al., 1994; Kainulainen et al., 1999). Further, essential hypertension is a multifactorial disease whereby many humoral factors may be involved in addition to the renin angiotensin system. It would be interesting to study ACE genetic polymorphism in the context of relatively isolated renin angiotensin system pathologies such as renal artery stenosis as a cause of secondary hypertension (Kotchen, 2009).

Conclusion

Although the findings are based on small figures and the sample is not representative of the Lebanese population, this study is a starting point for determining the association of ACE I/D polymorphism with hypertension in the Lebanese population. The significance of our findings is also limited by the fact that we did not analyze other potential risk factors such as body mass index and lifestyle and dietary factors, and this is due to incomplete data. It is recommended that more studies be conducted on larger samples collected from different geographical regions within Lebanon to further assess for ACE I/D polymorphism as a risk factor for the development of essential hypertension.

Footnotes

Acknowledgments

The authors are very grateful to Dr. Joseph Simaan for reviewing this manuscript. This study was funded by the Beirut Arab University.

Disclosure Statement

No competing financial interests exist.

References

Abbud

, Wilson

, Cosgrove

et al. 1998. Angiotensin-converting enzyme gene polymorphism in systemic hypertension. Am J Cardiol, 81:244-246.

Adrogue

, Madias

. 2007. Sodium and potassium in the pathogenesis of hypertension. N Engl J Med, 356:1966-1978.

Al-Eisa

, Haider

, Srivastva

. 2000. Angiotensin-converting enzyme gene insertion/deletion polymorphism and renal damage in childhood uropathies. Pediatr Int, 42:348-353.

Al-Eisa

, Haider

, Srivastva

. 2001. Angiotensin converting enzyme gene insertion/deletion polymorphism in idiopathic nephrotic syndrome in Kuwaiti Arab children. Scand J Urol Nephrol, 35:239-242.

Al-Gasham

, Ismail

, Dewaidar

et al. 2009. Methylenetetrahydrofolate reductase and angiotensin-converting enzyme gene polymorphisms among Saudi population from Qassim region. Genet Test Mol Biomarkers, 13:817-820.

Bayoumi

, Simsek

, Yahya

et al. 2006. Insertion-deletion polymorphism in the angiotensin-converting enzyme (ACE) gene among Sudanese, Somalis, Emiratis, and Omanis. Hum Biol, 78:103-108.

Bianchi

, Tripodi

, Casari

et al. 1994. Two point mutations within the adducin genes are involved in blood pressure variation. Proc Natl Acad Sci USA, 91:3999-4003.

Bouba

, Makrydimas

, Kalaitzidis

et al. 2003. Interaction between the polymorphisms of the renin-angiotensin system in preeclampsia. Eur J Obstet Gynecol Reprod Biol, 110:8-11.

Boulpaep

. 2003. Regulation of arterial pressure and cardiac output. Boron

, Boulpaep

. Medical Physiology. 1st. Saunders: Philadephia, 534-557.

10.

Chiang

, Chern

, Lai

et al. 1996. Age- and gender-dependent association of the angiotensin-converting enzyme gene with essential hypertension in a Chinese population. J Hum Hypertens, 10:823-826.

11.

Companioni

, Sautie

, Leal

et al. 2007. ACE I/D polymorphism study in a Cuban hypertensive population. Clin Chim Acta, 378:112-116.

12.

Das

, Pal

, Ghosh

. 2008. Angiotensin converting enzyme gene polymorphism (insertion/deletion) and hypertension in adult Asian Indians: a population-based study from Calcutta, India. Hum Biol, 80:303-312.

13.

El-Sibai

, Platt

, Haber

et al. 2009. Geographical structure of the Y-chromosomal genetic landscape of the Levant: a coastal-inland contrast. Ann Hum Genet, 73:568-581.

14.

Fatini

, Gensini

, Sticchi

et al. 2003. ACE DD genotype: an independent predisposition factor to venous thromboembolism. Eur J Clin Invest, 33:642-647.

15.

Frossard

, Obineche

, Elshahat

et al. 1997. Deletion polymorphism in the angiotensin-converting enzyme gene is not associated with hypertension in a Gulf Arab population. Clin Genet, 51:211-213.

16.

Gensini

, Padeletti

, Fatini

et al. 2003. Angiotensin-converting enzyme and endothelial nitric oxide synthase polymorphisms in patients with atrial fibrillation. Pacing Clin Electrophysiol, 26:295-298.

17.

Gesang

, Liu

, Cen

et al. 2002. Angiotensin-converting enzyme gene polymorphism and its association with essential hypertension in a Tibetan population. Hypertens Res, 25:481-485.

18.

Gonzalez-Ordonez

, Fernandez Carreira

, Medina Rodriguez

et al. 2000. Risk of venous thromboembolism associated with the insertion/deletion polymorphism in the angiotensin-converting enzyme gene. Blood Coagul Fibrinolysis, 11:485-490.

19.

Gunes

, Ata

, Degirmenci

et al. 2004. Frequency of angiotensin-converting enzyme gene polymorphism in Turkish hypertensive patients. Int J Clin Pract, 58:838-843.

20.

Higaki

, Baba

, Katsuya

et al. 2000. Deletion allele of angiotensin-converting enzyme gene increases risk of essential hypertension in Japanese men: the Suita Study. Circulation, 101:2060-2065.

21.

Hoffman

. 2007. Therapy of hypertension. Brunton

, Lazo

, Parker

. The Pharmacological Basis of Therapeutics. Mc Graw Hill: La Jolla, CA, 845-868.

22.

Hubert

, Houot

, Corvol

et al. 1991. Structure of the angiotensin I-converting enzyme gene. Two alternate promoters correspond to evolutionary steps of a duplicated gene. J Biol Chem, 266:15377-15383.

23.

Ismail

, Akhtar

, Nasir

et al. 2004. Association between the angiotensin-converting enzyme gene insertion/deletion polymorphism and essential hypertension in young Pakistani patients. J Biochem Mol Biol, 37:552-555.

24.

Jackson

, Brown

, Langdown

et al. 2000. Effect of the angiotensin-converting enzyme gene deletion polymorphism on the risk of venous thromboembolism. Br J Haematol, 111:562-564.

25.

Kainulainen

, Perola

, Terwilliger

et al. 1999. Evidence for involvement of the type 1 angiotensin II receptor locus in essential hypertension. Hypertension, 33:844-849.

26.

Kotchen

. 2009. Hypertensive vascular disease. Fauci

, Braunwald

, Kasper

et al. Harrison's Principles of Internal Medicine. www.accessmedicine.com/content.aspx?aID=2872428. 2009 January 5.

27.

Kunes

, Zicha

. 2009. The interaction of genetic and environmental factors in the etiology of hypertension. Physiol Res, 58:S33-S41.

28.

La Valle

, Issack

, Baitner

et al. 2001. The relationship of the factor V Leiden mutation or the deletion-deletion polymorphism of the angiotensin converting enzyme to postoperative thromboembolic events following total joint arthroplasty. BMC Musculoskelet Disord, 2:1.

29.

Marchlinski

. 2009. The tachyarrhythmias. Fauci

, Braunwald

, Kasper

et al. Harrison's Principles of Internal Medicine. www.accessmedicine.com/content.aspx?aID=2901807. 2009 January 5.

30.

Mastana

, Nunn

. 1997. Angiotensin-converting enzyme deletion polymorphism is associated with hypertension in a Sikh population. Hum Hered, 47:250-253.

31.

Morshed

, Khan

, Akhteruzzaman

. 2002. Association between angiotensin I-converting enzyme gene polymorphism and hypertension in selected individuals of the Bangladeshi population. J Biochem Mol Biol, 35:251-254.

32.

Philipp

, Dilley

, Saidi

et al. 1998. Deletion polymorphism in the angiotensin-converting enzyme gene as a thrombophilic risk factor after hip arthroplasty. Thromb Haemost, 80:869-873.

33.

Popov

, Fomicheva

, Kovalev

et al. 1996. Absence of association between the angiotensin-converting enzyme gene polymorphism and borderline hypertension in men of St Petersburg, Russia. J Hum Hypertens, 10:557-559.

34.

Ravn

, Benn

, Nordestgaard

et al. 2008. Angiotensinogen and ACE gene polymorphisms and risk of atrial fibrillation in the general population. Pharmacogen Genomics, 18:525-533.

35.

Rice

, Foy

, Grant

. 1999. Angiotensin converting enzyme and angiotensin II type 1-receptor gene polymorphisms and risk of ischaemic heart disease. Cardiovasc Res, 41:746-753.

36.

Rigat

, Hubert

, Henc-Gelas

et al. 1990. An insertion/deletion polymorphism in the angiotensin I-converting enzyme gene accounting for half the variance of serum enzyme levels. J Clin Invest, 86:1343-1346.

37.

Sabbagh

, Otrock

, Mahfoud

et al. 2007. Angiotensin-converting enzyme gene polymorphism and allele frequencies in the Lebanese population: prevalence and review of the literature. Mol Biol Rep, 34:47-52.

38.

Salem

, Batzer

. 2009. High frequency of the D allele of the angiotensin-converting enzyme gene in Arabic populations. BMC Res Notes, 2:99.

39.

Schmidt

, van

, Grobbee

et al. 1993. Polymorphism of the angiotensin I converting enzyme gene is apparently not related to high blood pressure: Dutch Hypertension and Offspring Study. J Hypertens, 11:345-348.

40.

Staessen

, Wang

, Ginocchio

et al. 1997. The deletion/insertion polymorphism of the angiotensin converting enzyme gene and cardiovascular-renal risk. J Hypertens, 15:1579-1592.

41.

Stanković

, Zivkovic

, Alavantic

. 2002. Angiotensin I-converting enzyme gene polymorphism in a Serbian population: a gender-specific association with hypertension. Scand J Clin Lab Invest, 62:469-475.

42.

Tohme

, Jurjus

, Estephan

. 2005. The prevalence of hypertension and its association with other cardiovascular disease risk factors in a representative sample of the Lebanese population. J Hum Hypertens, 19:861-868.

43.

Vassilikioti

, Doumas

, Douma

et al. 1996. Angiotensin converting enzyme gene polymorphism is not related to essential hypertension in a Greek population. Am J Hypertens, 9:700-702.

44.

Von Depka

, Czwalinna

, Wermes

et al. 2003. The deletion polymorphism in the angiotensin-converting enzyme gene is a moderate risk factor for venous thromboembolism. Thromb Haemost, 89:847-852.

45.

Zalloua

, Xue

, Khalife

et al. 2008. Y-chromosomal diversity in Lebanon is structured by recent historical events. Am J Hum Genet, 82:873-882.

46.

Zaman

, Yoshiike

, Date

et al. 2001. Angiotensin converting enzyme genetic polymorphism is not associated with hypertension in a cross-sectional sample of a Japanese population: the Shibata Study. J Hypertens, 19:47-53.