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European Journal of Heart Failure 2004 6(1):23-27; doi:10.1016/j.ejheart.2003.09.004
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© 2004 European Society of Cardiology

Association of the angiotensin-converting enzyme gene polymorphism with chronic heart failure in Chinese Han patients

Wenyan Huanga,*, Changqing Xieb, Hongyan Zhoua, Tianlun Yanga and Ming Suna

a Department of Cardiology, Xiang Ya Hospital Central South University, Changsha, Hunan, PR China
b Human Reproductive Engineering Lab, Xiang Ya Medical College Central South University, Changsha, Hunan, PR China

* Corresponding author. Tel.: +86-731-4804132. E-mail address: hwyswallow{at}163.com


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Background: An insertion/deletion (I/D) polymorphism is present in the 16th intron of the angiotensin-converting enzyme (ACE) gene and is associated with serum and tissue ACE level. Some studies have shown that the DD genotype is associated with some cardiovascular diseases; while ACE polymorphism's effect on chronic heart failure (CHF) remains uncertain.

Aim: To investigate the association of the ACE gene I/D polymorphism with CHF in the Chinese Han population.

Methods: The genotype was determined by polymerase chain reaction in 102 normal controls and in 79 patients with CHF. Plasma angiotensin (Ang) levels were assessed by radio-immunity assay. Left ventricular end-diastolic diameters (LVDD) and left ventricular ejection fractions were assessed by echocardiography.

Results: The ACE gene polymorphism distribution was similar in patients and control subjects. However, ACE gene DD polymorphism was associated with a more severe condition, greater LVDD [mm: DD: 71±7, ID: 62±5, II: 60±5, P<0.001 DD vs. ID, P<0.001 DD vs. II] and higher plasma Ang II level [pg/ml DD: 92±19, ID: 79±21, II: 65±17 P<0.05 DD vs. ID, P<0.001 DD vs. II].

Conclusion: In Chinese Han patients with CHF, ACE gene DD polymorphism might be a marker of a more severe condition, and a higher level of activation of the renin–angiotensin system.

Key Words: Heart failure • chronic • Peptidyl-dipeptidase A • Gene • Polymorphism

Received October 28, 2002; Revised March 26, 2003; Accepted September 25, 2003


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
The renin–angiotensin system (RAS) is a key molecular system in the pathogenesis of heart failure. Recently, a 287 base pair alu repeat sequence polymorphism of intron 16 of the angiotensin-converting enzyme (ACE) gene, which is associated with serum and tissue ACE levels [1,2], has been reported to correlate with some cardiovascular diseases [3,4]. However, the ACE polymorphism's effect on chronic heart failure (CHF) remains uncertain. Recently, this ACE gene polymorphism has been implicated in the pathophysiology of heart failure. A number of studies have suggested that the DD genotype may play a role in the severity of the decrease in left ventricular (LV) systolic performance, progressive LV dilation and increased mortality in patients with heart failure [5–7]. However, marked ethnic differences exist [8]. Limited data [9] has shown that there is no a similar relationship between the DD genotype and CHF in Chinese, which was attributed to the lower prevalence of the D allele in Chinese. We, therefore, conducted a study in Chinese patients to assess whether the ACE genotype is associated with CHF caused by systolic dysfunction.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
2.1. Patient and control group population
Between January 2001 and February 2002, we evaluated 79 consecutive, unrelated, Chinese patients with CHF as a result of idiopathic dilated cardiomyopathy (IDC), ischemic or hypertensive heart disease, or valvular heart disease, in our hospital. All patients had a left ventricular ejection fraction (LVEF) <=40%, as assessed by echocardiography. IDC was diagnosed if there was no obvious cause and an ejection fraction <=40%. Coronary angiography was performed in 57.8% (n=11) of the 19 patients with IDC. The remaining eight patients were all <45 years old with no risk factors for ischemic heart disease and no evidence of ischemia on exercise ECG testing. Patients with ischemic heart disease had a history of myocardial infarction (>6 months) or typical angina pectoris with risk factors and evidence of ischemia on exercise ECG testing or a positive result from coronary angiography. Patients were excluded from participation in the study if they had the following criteria: acute myocardial infarction, unstable angina pectoris, active rheumatic valve disease, severe valve regurgitation, significant hepatic or renal impairment or endocrine disease. Patients’ baseline data (including NYHA heart functional class, heart rate, blood pressure, serum sodium and creatinine) were obtained during the hospitalization before the treatment. A control group of 102 unrelated Chinese, who were free of cardiovascular disease, were recruited from those performing routine health examinations in our hospital. All subjects who participated in our study gave their informed consent. The investigation conforms to the principles outlined in the Declaration of Helsinki.

2.2. Echocardiographic methods
Left ventricular end-diastolic diameters (LVDD) were measured according to the American Society of Echocardiography guidelines [10] using a Hp5500 echocardiograph (USA) with a 2–4-MHz transducer attached. LVEF was calculated as previously described [11].

2.3. Hormonal measurements
Two milliliter venous blood samples were drawn from an antecubital vein after a 30 min supine rest in a fasting state between 8 and 9 AM. Samples were transferred to chilled disposable tubes containing 0.3 M 2-natrium-ethylenediamine tetra acetic acid 20 µl 0.34 M oxine 20 µl and 0.32 M ametoxin 10 µl. The disposable tubes were promptly centrifuged at 4 °C, and aliquots of plasma were immediately stored at –70 °C until analyzed. The levels of angiotensin (Ang) were measured by radio-immuno assay (North Biological Technology Research Institute, Beijing, China). The accuracy of angiotensin II determination is: sensitivity 10 pg/ml, range 10–2000 pg/ml, coefficient of variation between groups <15%, coefficient of variation within the group <10%.

2.4. ACE genotyping
Genomic DNA was isolated from whole blood (5 ml) by standard phenol–chloroform methods. The genotype of the ACE gene was determined by polymerase chain reaction (PCR) according to Rigat et al. [12]. Because the D allele in heterozygous samples was preferentially amplified, each sample found to have the DD genotype was subjected to a second, independent PCR amplification with a recently reported insertion-specific primer [13], which eliminated mistyping which could occur with the first system. The PCR products were resolved in 1.5% agarose gels and visualized with ethidium bromide staining.

2.5. Statistical analyses
All statistical analyses were conducted using the SPSS 10.0 statistical package. For continuous variables, comparisons among ACE genotypes or various conditions in patients were made using either an analysis of variance followed by a S–N–K/Dunnett's post hoc test or a Kruskal-Wallis H statistic, depending on whether or not variables were normally distributed (as determined from a one-sample Kolmogorov-Smirnov test for normal distribution) and whether the groups had equal standard deviations (as determined using Bartlett's test). Mann–Whitney U-test was used to assess differences in NYHA class between various conditions in patients. For categorical variables, comparisons among groups were performed by the {chi}2-test, in the subgroup comparisons the Brandt and Snedecor test was used. A K-means cluster analysis was used to divide the patients into three groups. A P value <0.05 was considered statistically significant.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
3.1. Frequencies of alleles and genotypes
The observed frequencies of the DD, ID and II genotypes in the control and patient groups were consistent with Hardy–Weinberg equilibrium. The genotype distribution and allele frequencies in the control group did not significantly differ from those in patient group (P>0.8) (Table 1) and were similar to those of previous reports [14,15] (P>0.1).


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  		Table 1 Distribution of the deletion (D)/insertion (I) polymorphism of the gene for ACE in the patients and control subjects

 
3.2. Characteristics of study population
The majority of patients were men (68.4%). The mean age of the cohort was 56±15 years. Most of the patients were in NYHA class III (40.5%) or IV (31.6%). The patients’ clinical characteristics by genotype are listed in Table 2. No significant differences in baseline data (age, sex, etiology, NYHA, heart rate, blood pressure, serum sodium and creatinine) were detected among the three-genotype subgroups.


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  		Table 2 Patient clinical characteristics by genotype

 
3.3. Associations between genotype and CHF
One-way ANOVA analysis was performed to assess the differences in clinical variables among the three genotypes. The only statistically significant differences were increases in LVDD (DD vs. ID, P<0.001; DD vs. II, P<0.001, according to post hoc statistical tests) and plasma Ang II levels (DD vs. ID, P<0.05; DD vs. II, P<0.001, according to post hoc statistical tests) in the DD group (Table 2). The patients were divided into three subgroups by a K-means cluster analysis according to the severity of their condition (mild, moderate or severe). Clinical characteristics (NYHA, LVDD, LVEF and Ang II levels) for each of these sub-groups are shown in Table 3. In subgroup 3, namely the severe subgroup, there was a higher frequency of DD genotype compared with that of II or ID genotype (P<0.001). The genotypic distribution in the three subgroups is given in Fig. 1.


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  		Table 3 Clinical characteristics of 79 patients with CHF divided according to severity

 


Figure 1
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  		Fig. 1 Genotypic distribution of CHF patients within different severity subgroups.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
In this study we found that distribution of the ACE gene polymorphism was similar in patients and control subjects. Patients homozygous for the deletion polymorphism showed an increase in LVDD and plasma Ang II levels compared to the other two patient genotypes. The ratio of patients with a severe condition to those with a mild/moderate condition was higher in DD genotype compared with that of II or ID genotype. Our results, therefore, suggest a possible role of ACE gene polymorphism in the progression of CHF, but not in the incidence of CHF.

In this study, there was a significant trend in the DD subjects, to a more severe condition (poorer NYHA functional class, lower LVEF, larger LVDD and higher plasma Ang II level) compared with that in the II/ID subjects. This significance was mainly caused by the increased plasma Ang II level and LVDD in the DD genotype subgroup, which might reflect a higher level of activation of the RAS and remodeling, and which may have led to the increased severity of CHF in the DD subjects. Since it has been confirmed that both LVDD [16] and plasma Ang II level [17] are independent predictors of mortality in CHF, it is possible that our results in a cross-section of patients with CHF reflect enhanced progression of the disease in patients with the DD genotype. Our results are consistent with those of Candy et al. [5], Raynolds et al. [6] and Andersson et al. [7]. The Candy study showed that the DD genotype of the ACE gene was independently associated with both a reduced LV systolic performance and an increased LV cavity size in patients with IDC. While in the latter two studies, they found a much higher prevalence of heart transplantation and mortality in DD homozygotes than those with the insertion sequence. The results of our study differ from that obtained by Sanderson et al. [9], who showed that ACE gene I/D polymorphism had no influence on severity in Chinese patients with CHF. However, the frequency of D allele reported by Sanderson et al. was 34.6% (patients), which is much lower than that determined in our study. In addition, the average age of their patients was older than ours; the negative result of the Sanderson study could thus be due to a decreasing power of the genetic with age [18].

We were unable to demonstrate an increased frequency of the ACE DD genotype in Chinese patients with CHF compared to controls. Although conflicting with some earlier studies, our result is consistent with a number of recent reports [19,20]. Overall, based on our data, we can speculate that in the Chinese Han population, the presence of ACE D allele does not increase the risk of myocardial injury, which initiates the heart failure syndrome, but instead may act as a disease modifier, altering the rate of disease progression.

4.1. Limitations
The study population contained patients with different causes of CHF. Although this may be considered a defect in the study and may serve to obscure any genetic effect, such a group of patients better reflects the real condition of CHF. Moreover, our conclusions are based on a population of 79 patients; any negative conclusion could thus be due to a low statistical power.

In summary, in Chinese Han patients with CHF, the frequency of ACE gene alleles does not differ from that of the general population, however, ACE gene DD polymorphism might be a marker of a more severe condition, and a higher level of activation of the RAS.


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 

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