Abstract
Aims
We investigated the correlation of adrenergic receptor polymorphisms, α2cDel322–325, β1Ser49Gly and β1Arg389Gly, with the risk of heart failure in the Japanese population.
Methods
These polymorphisms were analysed by polymerase chain reaction-restriction fragment length polymorphism in patients with chronic heart failure due to idiopathic dilated cardiomyopathy (DCM) and compared with the control group.
Results
There were no differences or any trends in the allele and genotype frequencies of the β1Ser49Gly and β1Arg389Gly polymorphisms. The allele frequency of the α2cDel322–325 variant was lower in patients than in controls (0.11 vs. 0.04, P = 0.011 < 0.017, by Bonferroni correction), while the genotype frequency just failed to reach significance (P = 0.022 > 0.017, by Bonferroni correction).
Conclusions
In this population, the variants β1Ser49, β1Arg389, and α2cDel322–325 do not appear to be risk factors for chronic heart failure due to DCM. The α2cDel322–325 variant may in fact confer some protection.
Keywords: adrenergic receptor, chronic heart failure, Japanese, polymorphism
Introduction
Neurohumoral factors play important roles in cardiac remodelling, determining the prognosis of heart failure. In particular, the sympathetic nervous system is activated in patients with chronic heart failure (CHF) [1] and sustained stimulation of the adrenergic system exerts direct adverse effects on cardiac function [2]. In spite of the importance of the adrenergic system, the effects of polymorphic mutation of adrenergic receptors on CHF remain to be fully elucidated.
In the present study, we focus on the presynaptic α2c adrenergic receptor (AR) polymorphism with the deletion of four consecutive amino acids, α2cDel322–325, and polymorphic amino acid variants of β1AR, Ser49Gly and Arg389Gly. These polymorphic changes result in alteration of AR function. The presynaptic α2AR negatively regulates the release of norepinephrine from cardiac sympathetic nerves [3] and α2cDel322–325 polymorphism shows a ‘loss-of-function’ phenotype [4]. The postsynaptic β1AR polymorphism, β1Ser49Gly, affects receptor sensitivity and promotes the downregulation of the receptor to agonists in vitro[5]. The change of β1AR from Arg to Gly at the 389 amino acid residue leads to the decrease in G-protein coupling [6]. Considering the importance of the adrenergic system as a modulator of cardiac remodelling, it could be proposed that polymorphisms of adrenergic receptor genes may be closely related to the risk of heart failure.
Recently, Small et al. proposed that the polymorphisms of β1Arg389Gly and α2cDel322–325 are synergistically related to the risk of CHF in a black population [7]. However several concerns, including the aetiology of heart failure and the absence of analysis of β1Ser49Gly frequency, have been raised against this study [8]. We have investigated the clinical significance of α2cDel322–325, β1Ser49Gly, and β1Arg389Gly for the risk of heart failure due to dilated cardiomyopathy (DCM) in the Japanese.
Methods
Subjects
The study subjects consisted of 91 unrelated consecutive patients with CHF due to idiopathic DCM (males 79.5%, age 58.4 ± 13.7 years, ejection fraction 34.6 ± 15.8%) who attended or were admitted to Hokkaido University Hospital, Kyoto Katsura Hospital, Osaka Prefectural Medical Centre for Respiratory and Allergic Diseases, Aizenbashi Hospital or Osaka City University Medical School Hospital. The ratio of the patients, classified as NYHA class I, II, III or IV, was 13.9, 46.8, 15.1, 24.1%, respectively. Patients with ischaemic cardiomyopathy were excluded. One hundred and nineteen subjects (all males, aged from 20 to 40 years) who had no history or symptoms of cardiovascular disease were chosen as controls. This study was approved by the institutional review committee. All subjects gave their informed consent to participate.
Genotyping
Genomic DNA was extracted from samples of peripheral blood leucocytes using the QIAamp DNA Blood Maxi Kit (Qiagen K.K., Tokyo, Japan) according to the manufacturer's protocol. Genotyping of α2cDel322–325, β1Ser49Gly and β1Arg389Gly polymorphisms was performed as described previously [4–6] with minor modifications.
Statistical analysis
Values were expressed as means ± SD. χ2 test of independence was used to test for associations between heart failure and allele. The 2 × 3 exact probability test was used to evaluate associations between heart failure and genotype. All of the analyses were corrected by Bonferroni correction. P < 0.017 was considered to be significant. Statistical analysis was performed with StatView Version 5.0 software (SAS Institute, Cary, NC, USA).
Results
The allele and genotype frequencies of α2cDel322–325, β1Ser49Gly and β1Arg389Gly polymorphisms in the patients and controls are shown in Table 1. The allele frequency of the α2cDel322–325 variant was lower in patients with CHF than in controls (0.04 vs. 0.11, P = 0.011 < 0.017, by Bonferroni correction) and the genotype frequency was not significant but showed the P-value nearly equal to the borderline of significance (P = 0.022 > 0.017, by Bonferroni correction). The allele and genotype frequencies of the β1Arg389 and β1Ser49 variants in the patients with heart failure were consistent with those of controls.
Table 1.
Distribution of α2c and β1 adrenergic receptor (AR) variants in controls and patients with heart failure
| Allele | ||||||
|---|---|---|---|---|---|---|
| Alleles and subjects | Frequency | P-value | Frequency | Genotype | P-value | |
| α2cDel322–325 | WT/WT | WT/Del | Del/Del | |||
| Controls | 0.11 | 0.011 | 95/119 (79.8%) | 23/119 (19.3%) | 1/119 (0.8%) | 0.022 |
| Patients with heart failure | 0.04 | 84/91 (92.3%) | 7/91 (7.7%) | 0/91 (0%) | ||
| β1Arg389 | Gly/Gly | Gly/Arg | Arg/Arg | |||
| Controls | 0.81 | 0.82 | 5/119 (4.2%) | 35/119 (29.4%) | 79/ 119 (66.4%) | 0.94 |
| Patients with heart failure | 0.80 | 5/91 (5.5%) | 26/91 (28.6%) | 60/91 (65.9%) | ||
| β1Ser49 | Gly/Gly | Gly/Ser | Ser/ Ser | |||
| Controls | 0.84 | 0.90 | 3/119 (2.5%) | 33/119 (27.7%) | 83/119 (69.7%) | 0.58 |
| Patients with heart failure | 0.84 | 4/91 (4.4%) | 21/91 (23.1%) | 66/91 (72.5%) | ||
P-values for comparisons of allele frequency or genotype frequency between controls and patients with heart failure were determined by 2 × 2 χ2 or by 2 × 3 exact probability test, respectively. P-value < 0.017 (0.05/3) was considered to be significant.
Combined genotypes of β1Arg389 and β1Ser49 variants were not associated with the risk of heart failure (Table 2).
Table 2.
Combined genotypes of β1AR and the risk for heart failure
| β1Ser49Gly | β1Arg389Gly | Controls | Patients with heart failure | Odds ratio for heart failure (95%CI) | P-value |
|---|---|---|---|---|---|
| No. of subjects | |||||
| 119 | 91 | ||||
| ≧ 1Gly | ≧ 1Gly | 6 | 6 | 1.00 | – |
| ≧ 1Gly | Arg/Arg | 30 | 19 | 0.63 (0.18–2.25) | 0.35 |
| Ser/Ser | ≧ 1Gly | 34 | 25 | 0.74 (0.21–2.55) | 0.43 |
| Ser/Ser | Arg/Arg | 49 | 41 | 0.84 (0.25–2.79) | 0.50 |
Subjects with at least one β1Gly49 allele and at least one β1Gly389 allele served as the reference group. Odds ratios and P-values between the reference group and each other group were determined by 2 ×2 χ2 test. P-value < 0.017 (0.05/3) was considered to be significant.
Discussion
The frequency of β1Arg389Gly and β1Ser49Gly polymorphisms did not differ from those in the control group, nor were there any trends, suggesting that these polymorphisms are not associated with susceptibility to CHF. Similarly, the combined genotype of β1Arg389Gly and β1Ser49Gly was not associated with the risk of CHF. However, we cannot definitely exclude the possibility that lack of association is derived from a β error problem, although the number of samples in our study was more than that in the previous study [7].
The allele frequency of the α2cDel322–325 variant was statistically lower in CHF than in the controls. However, it is uncertain that the α2cDel322–325 variant is a negative risk factor clinically, because the genotype frequency of this variant was of borderline significance, probably due to the low frequency of the homozygous genotype for α2cDel322–325 variant in the Japanese population. Thus, considering the limitation of low genotype frequency of this polymorphism, a reasonable interpretation of our results is that α2cDel322–325 variant is not a positive risk factor for CHF due to DCM in the Japanese population.
Previously, it was reported that allele frequency of α2cDel322–325 positively correlated with heart failure in both the white and black populations [7]. It remains to be clarified why our data are not consistent with the previous study [7]. One possibility is that the inconsistency might be derived from the difference in the cause of heart failure. The previous study included patients with ischaemic cardiomyopathy, while we excluded ischaemia because some adrenoceptor polymorphisms are related to hypertension, a risk factor for ischaemic heart disease [9, 10]. Another possibility is that the pathophysiological significance of the adrenoceptor polymorphism is closely related to the severity of heart failure. Importantly, the ratio of the patients classified as NYHA III or IV is lower in the present study than that in the previous report [7]. It may be hypothesized that there is a racial difference in the severity of CHF. Indeed, the previous study reported that Japanese patients with congestive heart failure show low mortality [11].
In addition to healthy controls, who are all males aged between 20 and 40 years, we analysed 189 diabetes patients (58% male with an age 60.4 ± 9.8 years) who did not suffer from CHF, as an age-matched control. It was found that there were no differences in the allele and genotype frequencies of α2cDel322–325 among healthy controls, male diabetes patients, and female diabetes patients (data not shown). Moreover, the allele frequency of α2cDel322–325 was lower in the patients with CHF than in those with diabetes (0.04 and 0.10, respectively). Thus it is unlikely that α2cDel322–325 polymorphism affected survival through other causes, resulting in the influence on allele frequency of samples.
Recent studies demonstrated that a genetic variability of β2AR, Thr164Ile, is closely related to heart failure. In patients with congestive cardiac failure, patients with homozygous genotype Ile/Ile show high mortality compared with those with other genotypes [12]. However, we could not statistically confirm the previous findings, probably because of low frequency of the mutation.
In summary, the α2cDel322–325, β1Ser49 and β1Arg389 variants do not appear to be risk factors for CHF due to DCM in a Japanese population, and the α2cDel322–325 variant may be protective. Considering the contradiction with the previous report, it is proposed that there may be a racial difference in the clinical importance of this polymorphism. Further efforts should be made to address any possible racial differences in the responsiveness of heart failure from different causes to β-blockers.
Acknowledgments
Shinji Negoro, Osamu Kato, Tomoyuki Hamaguchi and Isamu Yamamoto contributed to evaluation of chronic heart failure patients. Tsuyoshi Fukuda and Tomoko Kubota contributed to preparation of the manuscript. We thank Dr Mitsuru Sugawara (Department of Pharmacy, Hokkaido University Hospital) for collecting blood samples. We also thank Prof. Tatsuya Takagi (Department of Pharmaceutical Information Science, Graduate School of Pharmaceutical Sciences, Osaka University) for helpful discussion about statistics. This study was supported in part by a grant from the Organization for Pharmaceutical Safety and Research (OPSR) in Japan and Grants-in-Aid for Research on Measures for Intractable Diseases from Ministry of Health, Labour and Welfare and for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan. This study was also supported by a grant from Daiichi Pharmaceutical Co., Ltd.
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