Angiotensin-I Converting Enzyme Gene Polymorphisms and the ... · factors.1,2 Angiotensin-converting enzyme (ACE) has some biological effects that could be involved in the pathogenesis

Genetics

Angiotensin-I Converting Enzyme Gene

Polymorphisms and the Risk of Venous

Thromboembolism in an Ethnically Chinese

Population Living in Taiwan

Chien-An Hsieh,1 Yu-Lin Ko,2 Tsu-Shiu Hsu,1 Chi-Jen Chang,1 Ming-Sheng Teng,2 Semon Wu2 and Lung-An Hsu1

Background: There have been conflicting reports of the association between the insertion/deletion (I/D)

polymorphism of the angiotensin-converting enzyme (ACE) gene, and the risk of venous thromboembolism (VTE).

We sought to investigate the association between ACE I/D polymorphism, and the risk of VTE in a Chinese

population living in Taiwan.

Methods: 176 patients with VTE and 321 age and sex-matched controls were analyzed for the ACE I/D

polymorphism by polymerase chain reaction.

Results: The genotype distribution of the ACE I/D polymorphism was not statistically different between the VTE

affected subjects and the group of unaffected subjects (p = 0.057). Notably, the frequency of ACE D allele in patients with

VTE were significantly lower than that in the control group (28% vs. 35%, p = 0.018). After adjusting for age, gender,

smoking, hypertension, diabetes and body mass index (BMI), the ACE D allele carriers remained significantly associated

with a decreased risk of VTE. Further meta-analysis by pooling data from 15 studies revealed that neither the DD, nor the

II genotype, was found to be associated with VTE (pooled unadjusted odds ratio were 1.167, 95% confidence intervals,

0.927-1.470, p = 0.189 for DD, and 1.085, 95% confidence intervals, 0.875-1.345 p = 0.460 for II).

Conclusion: Our results suggest that the presence of the D allele may confer protection against the development of

VTE in an ethnically Chinese population in Taiwan. Further meta-analysis did not support a relationship between

the ACE I/D polymorphism and the risk of VTE.

Key Words: Angiotensin converting enzyme � Polymorphism � Genetics � Venous thromboembolism

INTRODUCTION

Venous thromboembolism (VTE) is a multifactorial

disease influenced by both genetic and environmental

factors.1,2 Angiotensin-converting enzyme (ACE) has

some biological effects that could be involved in the

pathogenesis of VTE, in that ACE converts angiotensin I

to angiotensin II, which is a potent vasoconstrictor and

can inhibit fibrinolysis, as well as enhance the activation

and aggregation of platelets.3 This enzyme also degrades

bradykinin, which is a powerful vasodilator and inflam-

matory mediator.4 Previous studies have suggested that

these factors are important in VTE pathogenesis,5,6 thus

Acta Cardiol Sin 2011;27:252�8 252

ACE Polymorphism and Venous ThromboembolismOriginal Article Acta Cardiol Sin 2011;27:252�8

Received: October 12, 2010 Accepted: March 3, 20111First Cardiovascular Division, Department of Internal Medicine,

Chang Gung Memorial Hospital, Chang Gung University College of

Medicine; 2The Division of Cardiology, Department of Internal

Medicine, Buddhist Tzu Chi General Hospital Taipei Branch, Taipei,

Tzu Chi University School of Medicine, Hualian, Taiwan.

Address correspondence and reprint requests to: Dr. Lung-An Hsu,

First Cardiovascular Division, Department of Internal Medicine,

Chang Gung Memorial Hospital, Chang Gung University College of

Medicine, No. 199, Tung-Hwa North Road, Taipei, Taiwan. Tel:

886-3-328-1200 ext. 8162, Fax: 886-3-327-1192, E-mail: hsula@

adm.cgmh.org.tw

implying that the serum ACE level may be associated

with the risk of developing VTE.

The ACE gene, located on chromosome 17q23, con-

tains an insertion/deletion (I/D) polymorphism in intron

16, which is characterized by the presence or absence of

a 287-bp fragment. This polymorphism accounts for

50% of the inter-individual serum ACE level variation,

with the DD genotype reported to have a higher serum

ACE level than the ID or II variants.7,8

Several studies previously investigated the relation-

ship between ACE I/D polymorphism and VTE,9-25 but

their findings have been controversial. This may have re-

sulted from ethnic differences between study popula-

tions. Among them, only one study was comprised of

Chinese subjects, and reported an association between

the DD genotype and the risk of pulmonary embolism.

Therefore, this study attempted to extend and confirm

their results for an ethnically Chinese population living

in Taiwan.

MATERIALS AND METHODS

Subjects

A total of 186 consecutive and unrelated Taiwanese

VTE patients, 87 males and 99 females, were referred to

the first cardiovascular division of Chang Gung Memo-

rial Hospital, Lin Kou, Taiwan, for venous Doppler

study. Those patients diagnosed with deep vein throm-

bosis (DVT) and/or pulmonary embolism were enrolled.

The mean age of VTE patients was 58.5 � 15.1 years.

Impedance plethysmography and ultrasonographic study

confirmed the diagnosis of deep vein thrombosis in all

such cases. Of the patients with VTE, 25 had pulmonary

embolism. The diagnosis of pulmonary embolism was

supported by one of the following confirmatory imaging

modalities: ventilation/perfusion radionuclide lung scan,

spiral pulmonary computed tomographic scan, or pulmo-

nary angiography. In 40 cases, VTE was associated with

cancer (n = 19), pregnancy (n = 5), oral contraceptives

(n = 1); or VTE occurred postoperatively (5 cardiac

catheterization, 10 surgery). In these cases, VTE was

classified as a secondary event. In the other 146 cases,

VTE was classified as a primary event. There were 360

control subjects, matched with VTE patients for age

(within two years) and sex, randomly selected from

among 718 subjects who underwent health examina-

tions, with no known history of major systemic and car-

diovascular diseases, including VTE, with mean age of

57.9 � 14.7 years, including 171 males and 189 females.

The demographic details of the VTE patients and their

control subjects have been described previously.26 Each

subject provided informed consent. The ethics commit-

tee at Chang Gung Memorial Hospital approved this

study.

Genomic DNA extraction and genotyping of

the ACE I/D polymorphism

Genomic DNA of patients and controls was isolated

from the peripheral blood leukocytes by standard me-

thod using proteinase K digestion of nuclei. The D and I

alleles were identified based on polymerase chain reac-

tion amplification (PCR) of the respective segments

from intron 16 of the ACE gene, as described pre-

viously.27 For quality control purposes, approximately

10% of the samples were re-genotyped using a blinded

procedure, with the same results. Further, in order to

confirm genotyping, a random sample of subjects was

re-genotyped by direct sequence analysis using a com-

mercial sequencing service and the primers previously

used for PCR amplification.

Statistical analysis

Clinical characteristics of continuous variables are

expressed as means � standard deviations (SDs), and

were tested using a two-sample t test. The chi-square test

was used to examine the differences in categorical vari-

ables, and to compare the allelic and genotype frequen-

cies. Odds ratios (OR) were calculated as a measure of

the association of the ACE I/D genotype with the VTE,

with the effects of the D allele assumed to be dominant

(which compares a combination of genotypes ID and DD

to the homozygous II), recessive (which compares a

combination of genotypes ID and II to the homozygous

DD), or additive (which compares a combination of

genotypes DD and ID with weights 2 and 1 respectively,

to the homozygous II). For each odds ratio, we calcu-

lated 2-tailed p values and 95% confidence intervals

(CI). A multivariable logistic regression analysis was

used to evaluate the independent effect of investigated

parameters on the risk of VTE. We used Metanalysis

software (version 1.2.0, 2004; Technopharma Srl, Italy)

253 Acta Cardiol Sin 2011;27:252�8

ACE Polymorphism and Venous Thromboembolism

for the meta-analysis. First, the within- and between-

study variation or heterogeneity was examined by Co-

chrane’s Q test. If a significant Q statistic (p < 0.05) in-

dicated heterogeneity across the studies, the DerSi-

monian and Laird method in the random effects model

was used for meta-analysis. Otherwise, the Mantel-

Haenszel method in the fixed effect model was selected.

Quantification of heterogeneity was also determined by

the I2 metric, which is independent of the number of

studies in the meta-analysis.28 Since not all studies per-

formed and reported the adjusted analysis, and there was

also no uniform set of confounders adjusted for by all

the studies, we used raw genotype data to yield pooled

unadjusted OR (which may be susceptible to confound-

ing). The sample size of our study could determine a

relative risk of > 38% for the D allele being a risk factor

for VTE with a power of 80% at an alpha level of 0.05

in a population with an incidence of D allele of 34%.

RESULTS

In this study, the genotyping of the ACE I/D poly-

morphism could not be determined in 10 cases in the

case group and 39 cases in the control group because of

a shortage of DNA sample. Table 1 lists the baseline

characteristics of the cases and control subjects. BMI

was significantly higher in VTE patients than in con-

trols. A substantially higher percentage of VTE patients

than controls were also diabetics. No significant devia-

tion from Hardy-Weinberg equilibrium was detected for

these two polymorphisms in either VTE patients or con-

trols (p = 0.34, and 0.62, respectively). The D allele fre-

quency of the ACE gene in the control group was 35%,

and consistent with findings obtained by our previous

works.27,29 The genotype frequencies in the study sam-

ples are shown in Table 2 according to whether the sub-

ject has VTE or not. The genotype distribution of the

ACE I/D polymorphism was not statistically different

between the VTE-affected subjects and the group of un-

affected subjects (p = 0.057). In contrast, the ACE D

allele frequencies of cases was significantly lower than

that in the control group (28% vs. 35%, p = 0.018, Table

2). As shown in Table 3, subjects carrying the ACE D

allele were significantly associated with a decreased risk

of VTE compared with those with homozygous II geno-

type in dominant and additive models. Although the

clinical profiles for the study population were incom-

plete, the association was also analyzed with a multi-

variable logistic regression analysis. After adjusting for

age, gender, smoking, hypertension, diabetes and BMI,

the ACE D allele carriers were significantly associated

with a decreased risk of VTE in all studied genetic

models (Table 3). Similar results were obtained when

cases with pulmonary embolism were compared to con-

trols (data not shown). Moreover, sub-classification of

VTE patients into primary or secondary subgroups

found no significant difference of the ACE genotype dis-

tribution when compared to controls (Table 2). Never-

theless, the ACE D allele carriers were still significantly


Chien-An Hsieh et al.

Table 1. Baseline characteristics of study population

Controls VTE patients p

Numbers 321 176

Sex (M/F) 149/172 81/95 0.933

Age, years 57.2 � 15.1 59.3 � 15.1 0.142

BMI, kg/m2 (n) 24.4 � 3.8 (309) 26.0 � 4.2 (139) < 0.001

Smoking, % (n) 33.2 (316) 29.1 (148) 0.369

Hypertension, % (n) 29.1 (316) 29.0 (155) 0.985

Diabetes, % (n) 06.6 (316) 12.3 (155) 0.040

* (n) represented case numbers without missing data; totally, 49

subjects had missing data on BMI status, 26 on hypertension

status, 33 on smoking status, and 26 subjects had missing data

on diabetes status.

Table 2. Genotype and allele frequencies of the ACE I/D polymorphism in the study population

Genotype Allele

II ID DDp

I D

Controls 138 (0.43)0 142 (0.44)0 41 (0.13) 0.65 0.35

VTE 95 (0.54) 65 (0.37) 16 (0.09) 0.057 0.72 0.28 0.018

Primary VTE 72 (0.52) 52 (0.38) 14 (0.10) 0.190 0.71 0.29 0.081

Secondary VTE 24 (0.60) 13 (0.33) 03 (0.07) 0.120 0.76 0.24 0.047

All p values are vs. controls.

associated with a decreased risk of VTE compared with

those with homozygous II genotype in secondary sub-

group (Table 2). Figure 1 shows the results of the meta-

analysis of our findings, together with 14 previous re-

ports.30 In total, this meta-analysis comprised 3624 cases

and 3812 controls. Cochrane’s Q test was statistically

significant (p < 0.001) and I2 was 77%, thus indicating

heterogeneity across the studies. Overall, the meta-

analysis demonstrated no statistically significant as-

sociation between the DD genotype and the risk of VTE

(DD versus ID + II, the pooled unadjusted OR = 1.167,

95% CI, 0.927-1.470, p = 0.189 by random effect). As

shown in Figure 2, similar results were obtained when

the effects of the D allele were assumed to be dominant

(which compares a combination of genotypes DD and ID

to the homozygous II) (pooled unadjusted OR = 1.085,



Table 3. Odds ratio (OR) and 95% confidence interval (CI) for venous thromboembolism (VTE) in subjects carrying the ACE I/D

polymorphism

model Genotype Control VTE OR (95% CI) p OR (95% CI)* p*

Dominant II 138 (43%) 95 (54%) 1.00 0.019 1.00 0.012

ID + DD 183 (57%) 81 (46%) 0.64 (0.44-0.93) 0.58 (0.37-0.89)

Recessive II + ID 280 (87.2%) 160 (90.9%) 1.00 0.218 1.00 0.02

DD 041 (12.8%) 16 (9.1%) 0.68 (0.37-1.26) 0.40 (0.18-0.92)

Additive II 138 (43%)0. 95 (54%) 1 0.020 1 0.003

ID 142 (44.2%) 065 (36.9%) 0.72 (0.54-0.95) 0.61 (0.44-0.86)

DD 041 (12.8%) 16 (9.1%)

* Multivariable logistic regression analysis with adjustments for age, gender, smoking, hypertension, diabetes and BMI.

Figure 1. Angiotensin-I Converting Enzyme (ACE) DD versus ID and II genotypes and the risk of venous thromboembolism (VTE).

95% CI, 0.875-1.345 p = 0.460; after excluding the

study of Della Valle et al.14 in which the numbers of ID

and II genotypes were not available). Since we have re-

ported that genetic variants in � fibrinogen (FGA) gene

were associated with susceptibility to VTE in this Tai-

wanese population,26 we further analyzed a possible

gene-gene interaction between the FGA T312A and ACE

I/D polymorphisms. We did not find a significant gene-

gene interaction (p = 0.946) between these two loci on

the risk of VTE.

DISCUSSION

This study analyzed the association between the

ACE gene I/D polymorphism and the susceptibility of

VTE in an ethnically Chinese population in Taiwan. Our

findings did not reproduce a previous report of associa-

tion between the ACE DD genotype and VTE on the

Chinese in China.15 In contrast, our results suggest that

the presence of the D allele may confer protection

against the development of VTE. Nevertheless, both

Lu’s15 and our studies revealed that the genotype distri-

bution of the ACE I/D polymorphism were not statisti-

cally different between the VTE-affected subjects and

the group of unaffected subjects. Furthermore, the sam-

ple size in this study is substantially larger than that in

the study of Lu et al.15

As shown in Figure 1, several studies have been

conducted to investigate the relationship between ACE

I/D polymorphism and VTE.9-25 Five of the 14 studies

independently demonstrated an association between the

ACE DD genotype and increased risk for VTE.10,13,15,18,19

In three studies, a protective effect of the DD genotype

against VTE was noted.11,20,23 The remaining six studies

did not have statistically significant results. There are

several possible reasons for the differences in the find-

ings of the various studies. These include ethnic hetero-

geneity of the ACE polymorphism, study design, the

broad spectrum of patients enrolled, artifacts of small

sample size, gene-gene and gene-environmental inter-

action.30

Observed association between the ACE D allele and

decreased risk of VTE is contrary to the common belief

and our initial expectation. Similar results have also

been reported in different Caucasian populations.11,20,23 The



Figure 2. ACE II versus ID and DD genotypes and the risk of VTE.

reasons, however, remain unclear. It could be due to

chance fluctuation, or other unknown gene-gene and

gene-environmental interactions. VTE is a multifactorial

disease influenced by genetic and environmental fac-

tors.1 Primary or idiopathic VTE was defined as a VTE

event without any triggering factors such as trauma,

surgery, immobilization, pregnancy, or the postpartum

period. It is possible that the presence of environmental

risk factors alters the association between ACE I/D poly-

morphism and VTE. Wells et al.20 postulated that the

ACE I/D polymorphism has a protective effect for idio-

pathic VTE, but this theory is weakened or even re-

versed if a transient condition is present (such as surgery

or cancer). Although our exploratory subgroup analysis

was underpowered, we did not find this gene and envi-

ronmental interaction. Nevertheless, the possibility that

ACE I/D polymorphism has differential effects in pa-

tients with primary versus secondary VTE still cannot be

excluded.

Our study has some limitations. First, the sample

size is relatively small. To overcome this limitation on

sample size, we incorporated our data into the pooled

data from a recent meta-analysis study by Hsiao and

Hsu,30 wherein the combined results still demonstrated

no association between VTE and either the DD or the II

genotype (the pooled unadjusted odds ratio was 1.167,

and 1.085, respectively). Second, there was a lack of in-

formation on serum ACE levels. Therefore, whether

ACE levels actually contribute directly to VTE cannot

be clearly determined. In addition, a positive association

between serum ACE levels and VTE has not yet been

established.30 Third, undiagnosed asymptomatic VTE in

the controls may be subject to some degree of error.

Other limitations included incomplete clinical profiles,

and lack of information for use of drugs such as ACE in-

hibitors and statins. Finally, because we did not assess

the influence of the ACE I/D polymorphism on the pre-

sence of other genetic thrombophilia factors with the

exception of FGA gene, we cannot exclude a possible

role of gene-gene interaction.

CONCLUSION

In conclusion, our results suggest that the presence

of the D allele may confer protection against the deve-

lopment of VTE to a limited extent, but further meta-

analysis does not support any association between the

ACE I/D polymorphism and the risk of VTE. A larger

prospective and longitudinal study, stratifying patients

by etiology and plasma ACE activity, would be neces-

sary to fully assess the significance of this polymor-

phism in the risk of VTE.

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Angiotensin-I Converting Enzyme Gene Polymorphisms and the ... · factors.1,2 Angiotensin-converting enzyme (ACE) has some biological effects that could be involved in the pathogenesis