Clinical and Experimental Hypertension, 31:471–480, 2009Copyright © Informa Healthcare USA, Inc.ISSN: 1064-1963 print / 1525-6006 onlineDOI: 10.1080/10641960902825479

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LCEH1064-19631525-6006Clinical and Experimental Hypertension, Vol. 31, No. 5, Jun 2009: pp. 0–0Clinical and Experimental Hypertension

ACE I/D and G2350A Polymorphisms in Pakistani Hypertensive Population of Punjab

ACE Polymorphisms and Hypertension in PakistanF.M. Alvi and S. Hasnain FARRAKH M. ALVI AND SHAHIDA HASNAIN

Department of Microbiology and Molecular Genetics, University of Punjab,Quaid-e-Azam Campus, Lahore, Pakistan

Several studies in different populations have shown mixed association of hypertensionwith ACE I/D and G2350A polymorphisms. To assess the link of these two polymor-phisms with hypertension in population of Punjab—most populated province of Paki-stan—we carried out this retrospective case control study in a mixed sample of 344hypertensive and normotensive controls. Genotype of the ACE I/D was determined bynested PCR and G2350A. polymorphism was determined by amplification of a smallfragment containing SNP and digesting it with the restriction enzyme. Statistical anal-ysis revealed that I/D polymorphism is not associated with hypertension in the Punjabipopulation c2 (df = 2) = 5.611, P £ 0.10. Overall, D allele frequency was 0.43 and Iallele frequency was 0.57. The G2350A polymorphism was found to be significantlyassociated with hypertension c2 (2df, c2 = 28.4, P £ 0.001). The G allele frequency(0.77) in our population was higher than previously reported. A combination of the AAand DD genotype seems to be linked with higher than average blood pressure levelboth in hypertensive and control groups. Our data suggests that the ACE I/D polymor-phism is not associated with hypertension but the G2350A polymorphism is associatedwith hypertension in the Punjabi population.

Keywords angiotensin-converting enzyme, essential hypertension, ACE I/D, G2350polymorphism, pakistani population

Introduction

Essential hypertension is very common but complex and a multigenic disorder amongworld populations. Onset of the disease depends upon demographic, environmental,vascular, and genetic factors (1). The angiotensin converting enzyme (ACE) is a keycomponent of rennin angiotensin aldosterone system (RAAS), which controls bloodpressure. The ACE breaks down inactive decapeptide angiotensin I to active vasoconstrictorangiotensin II. Another role of the ACE enzyme is to catabolize bradykinin (2), a strongvasodilator, to inactive bradykinin1-5. Angiotensin-converting enzyme inhibitors are

Submitted April 11, 2008; revised July 11, 2008; accepted July 30, 2008.Address correspondence to Shahida Hasnain, Department of Microbiology and Molecular

Genetics, Quaid-e-Azam Campus, University of Punjab, Lahore 54590, Pakistan; E-mail:genetic@brain.net.pk

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472 F.M. Alvi and S. Hasnain

among the most common group of anti-hypertensive drugs indicating involvement of theACE gene in hypertension. Serum ACE levels show variations among individuals (3).

Variations in plasma levels of the enzymes also suggest a possible link with thepolymorphisms in this gene. The ACE gene, located on chromosome 17, harbors an inser-tion/deletion polymorphism (I/D) in alu repeats of exon 16 which accounts for 47% of thevariance in plasma ACE concentration (4). Therefore, the ACE I/D polymorphism hasbeen widely studied to explore its role in hypertension and several other disease condi-tions (2). Most of the association studies have been reported in Chinese (5–8), Japanese(9,10), Mongolian (6,11), Pakistani (12), Bangladeshi (13), Tibetan (14), and Serbianpopulations (15). An absence of association was reported in Caucasian (16), Cuban (17),Japanese (18), and German populations (19). Conflict in the outcome of these studiesindicates that there may be some involvement of environmental or ethnic factors as well(20). Gene environment interaction is also indicated in a recent study on the Japanese malepopulation and according to it, a combination of II+ID genotype and daily salt intakeraises hypertension by an average of 10.5 mm Hg (21).

Another polymorphism in neighboring exon 17, ACE G2350A (dbSNP rs4343) wasfound to exert 19% effect on plasma ACE concentration (22). This polymorphism wasshown to be in strong linkage disequilibrium with hypertension by several researchers(4,22,23). This polymorphism has also been found to be associated with hypertension inthe Emirati population (24). Our aim was to look for the status of these polymorphisms inlocal populations and associate these polymorphisms with hypertension.

Methods

Study Group

The population under study consisted of 226 patients coming to Mayo Hospital Lahore—ateaching hospital linked with King Edward Medical University, Lahore. People comefrom all over Punjab province to this facility. Every patient was interviewed and consentwas taken on a history form designed for this purpose. The history form also containedinformation on blood chemistry indicating liver and kidney status. Another sample of 118persons coming to Mayo Hospital for reasons other than hypertension was also collectedin a similar fashion and acted as control. A study design was approved by the ResearchEthics Committee of the Punjab University.

From each patient, 5 ml of the venous blood was drawn into an EDTA containingtube and DNA was prepared using Genomic DNA isolation kits (Fermentas, Canada,Catalog No. K0512 and Promega, USA, Catalog No. A1125).

ACE I/D Genotyping

An ACE I/D polymorphism was detected by method of Rigat et al. (4) with minor modifi-cations. A polymerase chain reaction (PCR) with two specific forward primers (5′-CCCATC CTT TCT CCC ATT TCT C-3′) and (5′-GT TTC ACC GTT TTA GCC GGG A-3′)and one reverse primer (5′-CCA TGC CCA TAA CAG GTC TTC A-3′) was performedin a volume of 25 ul. The primers were slightly modified from Catherine, Eleanor, andNiall. (25) to achieve a similar annealing temperature. The reaction mixture contained 300ng DNA, 100 pM of each of three primers, and 2X PCR mix (Fermentas Catalog No.K0171) which comes premixed with Taq Polymerase, dNTPs, and a buffer-containingoptimum amount of magnesium chloride. Initial denaturation at 94°C for 5 min was

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ACE Polymorphisms and Hypertension in Pakistan 473

followed by 35 cycles of denaturation at 94°C for 30 s, annealing at 58°C for 40 s andchain elongation at 72°C for 1 min, followed by a final extension at 72°C for 3 min. Poly-merase chain reaction products were resolved on 2% agarose (Bio Rad, USA) gel andvisualized on Syngene’s Bio Imaging System (Gene Genius, Syngene, UK). In presenceof insertion (allele I), two bands 490 bp and 285 bp were seen, while a single 190 bp bandappeared in the case of deletion (allele D).

ACE G2350A Genotyping

Codon 2350 genotyping was done by a modified method of Saeed et al. (24) using primers(forward) 5′-CTG ACG AAT GTG ATG GCC GC-3′ and (reverse) 5′-TTG ATG AGTTCC ACG TAT TTC G-3′. The rest of the ingredients and PCR conditions were asdescribed by Saeed et al. (24). Polymerase chain reaction Product (5 ul) was digested with5 U of Bsh1236I (Fermentas, Canada, Catalog No. ER0921) for overnight, at 37°C toensure complete digestion. Digested fragments were separated on 10% acrylamide gel andidentified by ethidium bromide or by silver nitrate staining. A fragment of 122 bp afterdigestion corresponds to GG, and AA genotype was visualized as 100 and 22 bp frag-ments. Appearance of all three fragments was due to AG genotype.

Statistical Analysis

Clinical characteristics were expressed as mean ± SD. To compare the difference betweenmeans of hypertensive and normotensive characteristics, an unpaired t-test was used. TheHardy Weinberg equilibrium was checked using c2 test. Difference in genotype and allelefrequencies between the hypertension group and the control group were also examined byc2 analysis. The odds ratio was calculated using an online calculator (26). The interactionof the ACE I/D polymorphism with the G2350A polymorphism was studied by a chi-square test. A value of P < 0.05 was considered statistically significant unless otherwisestated.

Results

Demographic Information

Details of patients and controls included in this study are listed in Table 1. It shows thatthe ages of the control group were higher compared to the hypertensive group. Biochemi-cal parameters were estimated to make sure that patients were properly diagnosed foressential hypertension. Patients with a higher serum urea or a higher serum creatininelevel (showing malfunction of the kidneys) were excluded from the study. Similarly,diabetic patients were not included. Only those patients who were suffering from hyper-tension for at least 2 years and were already on medication were included in the study.

Genetic Analysis

Distribution of alleles and genotypes for two polymorphisms in hypertensive, as well as inthe control population, are listed in Table 2. Allele frequencies both in the control andhypertensive groups are in the Hardy Weinberg equilibrium for ACE I/D polymorphismbut differs in hypertensive patients for ACE G2350A (total c2 = 24.15, P ≤ 0.0001, malec2 = 14.92, P ≤ 0.0006, female c2 = 9.41, P ≤ 0.009).

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Tab

le 1

Cli

nica

l cha

ract

eris

tics

of

pati

ents

und

er s

tudy

Cha

ract

eris

tics

Tot

al N

umbe

r of

Cas

es (

345)

Mal

e (1

48)

Fem

ale

(197

)

HP

(226

)N

T (

119)

HP

(87)

NT

(61

)H

P (1

39)

NT

(58

)

Age

, y52

.8 ±

9.8

57.4

± 1

0.05

*54

.49

± 10

.20

60.4

2 ±

10.9

7**

52.0

7 ±

9.31

54.3

4 ±

7.97

SBP,

mm

Hg

152.

44 ±

12.3

711

6.24

± 1

3.24

*15

5.29

± 1

1.36

118.

33 ±

14.

98*

150.

88 ±

12.

5611

4.29

± 1

0.76

*D

BP,

mm

Hg

92.4

9 ±

6.72

73.5

5 ±

6.13

*94

.30

± 6.

5674

.00

± 5.

73*

91.5

7 ±

6.58

73.2

1 ±

6.50

*U

rea,

mm

ol/L

5.20

± 1

.34

4.98

± 1

.38

5.10

± 1

.65

4.68

± 1

.77

4.60

± 1

.44

4.33

± 1

.52

Cre

atin

ine

umol

/L87

.76

± 8.

0587

.39 ±

8.55

***

88.4

5 ±

8.32

87.6

7 ±

8.5

87.0

7 ±

7.8

87.1

2 ±

8.6

Suga

r m

mol

/L4.

74 ±

0.3

74.

69 ±

0.4

24.

72 ±

0.5

14.

66 ±

0.4

84.

27 ±

0.4

74.

01 ±

0.5

3***

*

Val

ues

are

mea

n ±

SD.

Abb

revi

atio

ns: H

P-h

yper

tens

ive;

NT

-nor

mot

ensi

ve;

SBP

-sys

toli

c bl

ood

pres

sure

; D

BP

-dia

stol

ic b

lood

pre

ssur

e.*P

< 0

.000

1; *

*P <

0.0

010;

***

P <

0.0

015;

***

*P <

0.0

384

(unp

aire

d t-

test

).

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Tab

le 2

Dis

trib

utio

n of

gen

otyp

es a

nd a

llel

es f

or A

CE

I/D

and

G23

50A

pol

ymor

phis

ms

in h

yper

tens

ive

and

norm

oten

sive

pop

ulat

ion

Phen

otyp

e/G

enot

ype

HP

Subj

ects

NT

Sub

ject

sc2

OR

(95

% C

I) H

P vs

. NT

AC

E I

/D5.

611

(df

= 2

, P ≤

0.1

0)D

D46

(0.

20)

16 (

0.14

)1.

63 (

0.88

–3.0

2, P

= 0

.62)

ID10

2 (0

.45)

47 (

0.40

)1.

24 (

0.79

–1.9

5 P

= 0

.55)

II78

(0.

35)

55 (

0.47

)0.

60 (

0.38

–0.9

5 P

= 0

.375

)c2

(fo

r H

WE

)1.

406

(P≤

0.49

51)

1.31

8 (P

≤ 0

.514

7)D

0.43

0.34

2.63

3 (P

≤ 0

.104

7)I

0.57

0.67

AC

E G

2350

AG

G15

1 (0

.67)

47 (

0.40

)28

.4 (

df =

2, P

≤ 0

.001

)3.

04 (

1.92

–4.8

2, P

= 0

.75)

AG

48 (

0.21

)57

(0.

48)

0.29

(0.

18–0

.47,

P =

0.2

2)A

A27

(0.

12)

14 (

0.12

)1.

01 (

0.50

7–2.

00, P

= 0

.50)

c2 (

for

HW

E)

24.1

5* (

P ≤

0.00

01)

0.27

3 (P

≤ 0

.872

4)G

0.77

0.64

6.18

(df =

1, P

≤ 0

.012

9)A

0.23

0.36

Abb

revi

atio

ns: H

WE

-Har

dy W

einb

erg

equi

libri

um. O

ther

abb

revi

atio

ns a

re s

ame

as s

how

n in

Tab

le 1

.*N

ot in

HW

E.

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476 F.M. Alvi and S. Hasnain

Association of three ACE I/D genotypes (DD, II, and ID) with essential hypertensionwas not found to be significant (total hypertensive vs. total normotensive: c2 = 5.61, 2 df,P < 0.10) in our sample. The DD genotype was related to hypertension (odds ratio of 1.63)as compared to II (odds ratio 0.60), but this difference was not statistically significant.

The association of ACE G2350A genotypes with the clinical phenotype were significantc2 = 28.4, 2 df, P < 0.001). The odds of GG genotypes were 3.04 times more in hypertensivepatients (95% CI = 1.92–4.82, P = 0.75) compared to normotensive patients (Table 2).

We also evaluated the combined effect of the four alleles through construction ofhaplotypes on hypertension. Table 3 shows the distribution of haplotypes in cases andcontrols. The haplotypes frequency of I/G and D/G were higher in the hypertensive groupcompared to control group.

Discussion

The population of the Punjab represents a genetically unique population due to in-breeding forcenturies. People prefer to marry within cast or to close relatives (mostly first cousins).For this study, control individuals were carefully selected and had neither a personal norfamily history of hypertension. Since it could not be predicted that individuals of thecontrol group would develop hypertension later on, the age of the control group was set at> 50 years (average 57.4 years) to minimize sampling error.

Variation in plasma ACE levels among individuals (3) and linkage of the ACEpolymorphism with variations in the serum enzyme level (4) have forced the researchersto focus on the ACE gene and its polymorphisms. European and African populations havebeen shown to be in strong disequilibrium with the 16 kb region of the ACE gene (21).Furthermore, the presence of quantitative trait locus (QTL) in the ACE gene influencingACE levels in serum suggests links with either the ACE I/D polymorphism or with someother nearby locus (27,28). We found no difference in genotypes of I/D polymorphismbetween hypertensive and normotensive population. The D allele was more prevalent inthe hypertensive population (43%) compared to the normotensive (34%) population. TheII genotype was higher in the normotensive (67%) population compared to the hyperten-sive population (57%). The I allele was more common in males compared to females inthe control group as well as the hypertensive group. The I/D polymorphism has been stud-ied in several populations and in several diseases with variable results (6–15). In a sampleof 211 hypertensive patients of the Islamabad region of Pakistan, I allele was also higher(55%) than D alleles (45%) but no association with hypertension was indicated (12). Similarly,no association was found in Chinese populations of Han and Dongxiang regions (7).Forty-three percent of D allele frequency is lower than previously reported but higher thanthe Japanese population (18).

Table 3Haplotypes frequency of the ACE I/D and G2350A polymorphisms

Haplotypes Hypertension Controls OR (95% CI) P-Value

I/G (216) 0.48 (89) 0.37 1.545 (1.121–2.130) 0.607D/G (165) 0.37 (64) 0.27 1.574 (1.11–2.22) 0.611D/A (21) 0.05 (17) 0.07 0.636 (0.329–1.231) 0.389I/A (48) 0.11 (68) 0.29 0.298 (0.198–0.450) 0.230

Values indicate frequency of haplotypes. c2 (4x2) = 39.75, df = 3, P ≤ 0.001.

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ACE Polymorphisms and Hypertension in Pakistan 477

Out of 13 polymorphisms reported by Zhu et al. polymorphism in exon 17, ACEG2350A had the most significant effect on the plasma ACE level after I/D polymor-phism (22). Our study shows a significant association of G2350A polymorphism withhypertension in the Punjabi population. These results are consistent with Saeed et al.(24) but in contrast to a recent study in a Japanese population, where no direct associa-tion has been found with hypertension but a significant association was found with leftventricular hypertrophy (29). Genotype GG was found to be 67% in hypertensivegroups higher than the control group (40%). Among hypertensive patients, males have ahigher percentage of GG genotype compared to female patients. Overall, G allelefrequency (77%) in both groups was somewhat higher then previously reported G = 0.70(30,31). The G2350A genotype in hypertensive patients did not occur in the HardyWeinberg equilibrium in our study group. The Punjabi population is a relatively closeand homogenous population having a high degree of consanguinity. The G allele mayhave pooled in our population; an increase in sample size may provide a more accuratepicture. The genotype in the control group was in agreement with the Hardy Weinbergequilibrium.

Figure 1. Graphs showing average diastolic and systolic blood pressures of patients with combinedgenotypes of two polymorphisms—ACE G2350A and I/D. Distribution in total sample is shown as(A) and (B). Results of males are shown in (C) and (D), while (E) and (F) show blood pressures infemales. Abbreviations: SBP-systolic blood pressure; DBP-diastolic blood pressure (measured inmm Hg); HP-hypertension group; NT-normotensive group

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478 F.M. Alvi and S. Hasnain

The influence of a combination of genotypes of I/D and G2350A polymorphisms onsystolic and diastolic blood pressure was investigated by counting the number of individu-als with a particular genotype (e.g., DD and AA) and counting their average systolic anddiastolic blood pressure as shown in Figure 1. The DD allele has been reported to be asso-ciated with diastolic blood pressure in the Argentina population (32). Interestingly, a com-bination of DD and AA genotypes seems to be linked with higher systolic and diastolicblood pressure values in both hypertensive and normotensive groups (Figure 1) and thiscombination is more pronounced in males compared to females.

Haplotype construction revealed that among the possible haplotypes, I/G was the mostfrequent (OR 1.545). The D/G combination is also significantly higher in the hypertensivegroup (OR 1.574). These results are similar to that found in the Emirati population (31). Geneinteraction studies give much more insight into the understanding of the complex genetic dis-eases as indicated by a recent study in the Korean population where interaction betweenGNB3, C825T, and ACE I/D polymorphisms was linked to essential hypertension (33).

Case control studies are extensively applied to search for genomic loci that may con-tribute to the onset of genetic diseases. Despite being low in cost and an easy collection ofthe cases, case control studies can lead to false-positive associations unless confirmed byother studies in different populations. Because of the complex nature of the disease, wesuggest that further studies with a combined effect of several SNPs be carried out. Ourdata supports the association of the G2350 polymorphism with hypertension but does notsupport the association of hypertension with the ACE I/D polymorphism.

Declaration of Interest

The authors report no conflicts of interest. The authors alone are responsible for thecontent and writing of the paper.

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