Role of ß1- and ß2-adrenoceptor polymorphisms in heart failure: a case-control study

Loredana Covoloa, Umberto Gelattia, Marco Metrab,*, Savina Nodarib, Antonio Piccichèa, Natalia Pezzalib, Claudia Zania, Adriana Albertia, Francesco Donatoa, Giuseppe Nardia and Livio Dei Casb

a Cattedra di Igiene, Università   di Brescia, Italy
b Cattedra di Cardiologia, Università  di Brescia, c/o Spedali Civili, P.zza Spedali Civili 1, 25123 Brescia, Italy

Received November 4, 2003; revised May 30, 2004; accepted June 10, 2004 * Corresponding author. Tel.: +39-30-3995572; fax: +39-30-3700359 (E-mail: metramarco{at}libero.it).


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Conflict of interest
 References
 
BACKGROUND: We hypothesised that the polymorphisms of the genes encoding for ß1- and the ß2-adrenoceptors may have a role in the pathogenesis of heart failure (HF). We therefore compared the polymorphisms of the ß1-adrenoceptor gene (Arg389Gly), the ß2-adrenoceptor gene (Arg16Gly, Gln27Glu) and their combinations in patients with HF and normal subjects living in the same area.

METHODS AND RESULTS: A total of 256 cases with HF (left ventricular ejection fraction ⩽40%) and 230 normal subjects were enrolled. The ß1- and ß2-adrenoceptor gene polymorphisms were assessed by PCR, followed by restriction enzyme digestion. No differences were observed in the distribution of any of the three genotypes studied in patients with HF and normal subjects. An analysis of the genotype combinations showed a non-significant increase in the risk of HF associated with the Arg389Gly16Gln27 (odds ratio=1.4; 95%CI 0.5–3.6) and Arg389Gly16 Glu27 (odds ratio=1.2; 95%CI, 0.5–2.8) homozygous allele combinations.

CONCLUSION: None of the three most common polymorphisms of ß-adrenoreceptors are associated with an increased risk of HF.


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Conflict of interest
 References
 
In the last few years, a decrease in the mortality and morbidity of the main cardiovascular diseases has coincided with a progressive increase in the incidence, prevalence and mortality of heart failure (HF).1–3 Furthermore, the economic impact of HF on health services is increasing due to the long-term pharmacological treatment and the frequent hospitalisation of these patients.4 It is therefore important to identify the risk factors for HF so that preventive measurements can be undertaken early, before the onset of overt clinical symptoms.5

Sympatho-adrenergic activation is known to play a pivotal role in the progression of HF.6,7 We hypothesised that the polymorphisms associated with a different adrenergic receptor (AR) sensitivity to the sympathetic signal may play a role in the risk of HF. Both ß1- and ß2-ARs genes are polymorphic.8 With regard to the ß1-AR gene, a single nucleotide polymorphism (SNP) resulting in an arginine (Arg) to glycine (Gly) switch at amino acid 389 (Arg389Gly) is located at the carboxy terminus of the receptor near the seventh transmembrane region9 (Fig. 1). Previous in vitro studies have shown that Arg389 receptors have 3-fold higher maximal isoproterenol-stimulated levels of adenylate cyclase activities compared to Gly389 polymorphic receptors.10 Therefore, Arg389 homozygotes may be more exposed to the untoward effects of catecholamines.



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Fig. 1 Schematic description of ß1-adrenoceptor with the location of polymorphic site and relative phenotype. CM: Cell membrane; I: Intracellular; E: Extracellular.

 
Although the adverse effects of sympathetic stimulation are mainly mediated by ß1-ARs, ß2-ARs may also be important.6,7,11 However, they may also have favourable effects as they may be associated with an increased exercise capacity12,13 and may inhibit apoptosis 14 and Gαq-mediated hypertrophy15 in the myocardium. Their role is further increased by concomitant ß1-AR downregulation.6,11 Several SNPs have been described in the gene encoding the human ß2-AR. The most frequent polymorphisms are located in the amino-terminal region of the receptor and consist of arginine (Arg) to glycine (Gly) substitution at position 16 (Arg16Gly) and glutamine (Gln) to glutamic acid (Glu) substitution at position 27 (Gln27Glu)16 (Fig. 2). An in vitro study17 has shown that the Gly16 allele is associated with enhanced agonist-induced downregulation. On the contrary, Dishy et al.18 found that the Gly16 allele was associated with resistance to desensitisation in an in vivo model. Data on the Gln27Glu polymorphism are not controversial. The Glu27 allele is associated with resistance to downregulation, relative to the responses of the wild-type allele.13,16–18 There is marked linkage disequilibrium between these polymorphisms. Almost all subjects who are homozygous for Glu 27 are also homozygous for Gly 16, whereas those homozygous for Gly 16 can be homozygous for Gln 27, homozygous for Glu 27, or heterozygous at codon 27.19 For this reason, both in vitro and in vivo studies must also consider the haplotype or combinations of more than one SNP rather than single-point mutations.



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Fig. 2 Schematic description of ß2-adrenoceptor with the location of polymorphic sites and relative phenotype. CM: Cell membrane; I: Intracellular; E: Extracellular.

 
Despite the pathogenetic importance of adrenergic stimulation, the relationships between ß-AR genetic polymorphisms and cardiovascular disease are still unclear.20–25 We therefore investigated the possible association of ß1-AR and ß2-AR gene polymorphisms and HF using a case-control study design.


    Methods
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 Conflict of interest
 References
 
Patients
A total of 256 consecutive subjects admitted to the Institute of Cardiology of the University and Spedali Civili of Brescia, North Italy, with a diagnosis of HF were enrolled as cases between January and December 2002. Only cases who were born in Italy, Caucasian and with HF due to coronary heart disease (CHD) or idiopathic dilated cardiomyopathy (IDC) were included. The diagnosis of HF was based on the presence of the typical clinical signs and symptoms of HF with a left ventricular ejection fraction (LVEF)⩽40% at two-dimensional echocardiography.26,27

A random sample of 230 subjects from the general population living in the same area, all Caucasian, were enrolled as controls. Subjects were considered eligible as controls if they were of the same age range as the cases (30–80 years), born in Italy and Caucasian, and with no clinical symptoms or signs suggesting the presence of HF.26,27 The controls underwent clinical and echocardiographic evaluation by a cardiologist to detect the presence of HF, left ventricular dysfunction or valve disease even if asymptomatic. Subjects with symptoms or signs that could be related to HF or with an abnormal echo-Doppler exam were excluded.

A 20 ml blood sample was taken by venipuncture from all the subjects and the serum was stored at –80 °C until further analysis. A local Ethics Committee approved the project and written informed consent was obtained from all the subjects.

Genotyping
Genomic DNA was extracted from 200 μl of EDTA anti-coagulated blood using a QIAamp DNA Blood Mini Kit (Qiagen s.p.A.).

Genotyping was performed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). The samples were analysed for Arg389Gly of ß1-AR gene as previously described.18 The amplification reaction was carried out in a final volume of 50 μl containing 300–500 ng DNA, 0.02 mM of each deoxynucleotide triphosphate, 10% buffer (100 mM Tris–HCl, 15 mM MgCl2, 500 mM KCl, pH 8.3), 5% DMSO and 2.5 U Taq DNA polymerase. The primers were 5'-TGGGCTACGCCAACTCGG-3' and 5'-GGCCCCGACGACATCGTC-3'. The DNA was amplified for 30 cycles with denaturation at 94 °C, annealed at 59 °C, with extension at 72 °C for 1 min at each step using a PCR Express thermal cycler (Celbio S.r.l., Milan, Italy). PCR products were analysed by 2% agarose gel electrophoresis (NuSieve 3:1, BMA, Rockland, ME, USA) and visualised by ethidium bromide staining. For polymorphism analysis the amplified products were digested at 60 °C for 1 h with 10 U of BstN I (BioLabs, Celbio S.r.l., Milan, Italy). The fragments were resolved on a 2.5% ultra-pure DNA agarose gel electrophoresis (NuSieve 3:1, BMA, Rockland, ME, USA) and visualised under ultraviolet illumination by ethidium bromide staining. This digestion produced fragments of the following sizes: 221 bp in Arg389 homozygotes; 67, 154 and 221 bp in heterozygotes; 67 and 154 in Gly389 homozygotes. The two polymorphisms in ßAR-2 gene which result in Arg16Gly and Gln27Glu were analysed as previously described.25 Amplification reaction was carried out in a final volume of 26 μl containing 300–500 ng DNA, 0.38 mM of each deoxynucleotide triphosphate, 10% buffer (100 mM Tris–HCl, 15 mM MgCl2, 500 mM KCl, pH 8.3) and 1 U Taq DNA polymerase.

Amplification of the DNA segment containing codon 27 of the ß2-AR gene had as the forward primer 5'-GGCCCATGACCAGATCAGCA-3' and as reverse primer 5'-GAATGAGGCTTCCAGGCGTC-3'.

Amplification of the DNA segment containing codon 16 of the ß2-AR gene was carried out the same way as for codon 27 but using 5'-CTTCTTGCTGGCACGCAAT-3' as the forward primer and 5'-CCAGTGAAGTGATGAAGTAGTTGG-3' as reverse primer. The DNA was amplified for 30 cycles with denaturation at 94 °C for 1 min, and annealed at 63 °C for codon 27, at 56 °C for codon 16, with extension at 72 °C for 1 min using a PCR Express thermal cycler (Celbio S.r.l., Milan, Italy). PCR products were analysed by 2% agarose gel electrophoresis (NuSieve 3:1, BMA, Rockland, ME, USA) and visualised by ethidium bromide staining.

For detection of the Gln27Glu polymorphism the amplified products were digested at 37 °C for 1 h with 2.5 U of Fnu4H I. This digestion produced fragments of the following sizes: 27, 55, 97 and 174 bp in Gln27 homozygotes; 27, 55, 97, 174 and 229 bp in Gln27Glu27 heterozygotes; and 27, 97 and 229 bp in Glu27 homozygotes. The 27 bp fragment was too small to be resolved on the gel.

For detection of the Arg16Gly polymorphism the amplified products was digested at 60 °C for 1 h with 1 U of BsrD I. This digestion produced fragments of the following sizes: 14, 56 and 131 bp in Arg16 homozygotes; 14, 23, 56, 108 and 131 bp in Arg16Gly16 heterozygotes; and 14, 23, 56 and 108 bp in Gly16 homozygotes. The fragments were resolved on a 2.5% ultra-pure DNA agarose gel electrophoresis (NuSieve 3:1, BMA, Rockland, ME USA) and visualised under ultraviolet illumination by ethidium bromide staining.

Statistical analysis
In this study, we analysed the risk of HF only for genotypes with >10% prevalence. We therefore calculated that a sample size of at least 230 cases and 230 controls was necessary to have a power of 80%, with an alpha error of 0.05 and a two sided test, to obtain an odds ratio of 2 for a SNP having a prevalence of 15% in the general population.

Another 26 cases were enrolled to further increase precision in the prevalence estimates in the diseased people. Therefore, the analysis was performed on a frequency-matching and not individual matching design, according to commonly used methods for epidemiological research.28

Differences in clinical variables and in the distribution of ßAR-1 and ßAR-2 genotypes among cases and controls were assessed using the unpaired t-test, {chi}2 and exact test analysis. In order to test for the effect of each genotype and their interaction the odds ratios (ORs) and their 95% confidence intervals (95% CI) were computed by unconditional logistic regression analysis using the maximum likelihood method. Subjects' sex and age were included in the logistic regression models as possible confounders.

It was preferred not to perform formal statistical tests to highlight the OR estimates since a single CI can be much more informative than a single p-value, according to currently used methods in epidemiological research.29 It should be noted, however that the 95% CIs of the OR estimates cannot be interpreted properly as the results of statistical tests due to the many estimates performed in the study.

To test for Hardy–Weinberg equilibrium for each polymorphism, the expected genotype numbers were calculated from the allele frequencies, and deviation from the observed genotype numbers was determined using the {chi}2-test. All the analyses were computed two-sided using the STATA statistical package (Stata Statistical Software: Release 7.0. College Station, TX: Stata Corporation).


    Results
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 Conflict of interest
 References
 
A total of 256 HF cases and 230 controls were enrolled. The distribution of cases and controls by sex, age and presence of the main risk factors for HF is shown in Table 1. There were no differences between cases and controls with regard to the presence of risk factors for HF, except for diabetes which was present in 24.2% of cases and 7.8% of controls (p<0.05).


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Table 1. Characteristics of cases and controls
 
Table 2 shows the distribution and the odds ratios for HF of the polymorphisms of the ß1-AR (codon 389) and 2-AR (codon 16 and codon 27). The frequency of subjects homozygous for the Arg389 allele was not significantly different between the cases and the controls (46.5% versus 53.1%). With regard to ß2-AR, the frequency of subjects homozygous for the Gly16 allele was 37.9% in cases and 35.2% in controls, and the frequency of subjects homozygous for the Glu27 allele was 13.7% in cases and 13.5% in controls. No increase of ORs was observed for each polymorphism considered. There was no deviation from the Hardy–Weinberg equilibrium for any of the polymorphisms considered when comparing expected and observed genotype frequencies for both cases and controls. No significant association was found when the patients were split according to their aetiology, either (Table 3).


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Table 2. Odds ratio (ORs) for heart failure adjusted for age and sex and 95% confidence intervals (95% CI) according to ß1- and ß2-adrenoceptor genotypes
 

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Table 3. Odds ratio (ORs) for coronary heart disease (CHD) and idiopathic dilated cardiomyopathy (IDC) adjusted for age and sex and 95% confidence intervals (95% CI) according to ß1- and ß2-adrenoceptor genotypes
 
The haplotype analysis showed no significant difference with regard to the prevalence of the Gly16Glu27, Gly16Gln27 and Arg16Gln27 genotypes in HF patients compared with normal subjects (Table 4). Finally, we analysed the haplotypes Gly16Gln27, Gly16Glu27 and Arg16Gln27 in combination with the Arg389 variant of the ß1-AR gene (Arg389Gly16Gln27, Arg389Gly16Glu27 and Arg389Arg16Gln27). The ORs for HF of these genotype combinations are shown in Table 5. A non-significant increase in HF risk was observed among subjects carrying the Arg389Gly16Gln27 and the Arg389Gly16Glu27 genotypes (OR=1.4, 95% CI: 0.5–3.6 and OR=1.2, 95% CI: 0.5–2.8, respectively). Significant interactions were neither found when these analyses were repeated with the cases split according to either sex or HF aetiology (data not shown).


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Table 4. Odds ratios for heart failure adjusted for age and sex according to allele combinations of ß2-AR genes
 

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Table 5. Odds ratios for heart failure adjusted for age and sex according to allele combinations of ß1- and ß2-AR genes
 

    Discussion
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 Conflict of interest
 References
 
Increased ß-adrenergic receptors stimulation has detrimental effects to the heart and is a major cause of the progression of HF.6,7,11,30 ß1- and ß2-AR polymorphisms may significantly influence the sensitivity and density of ß-ARs.10,16,17 We therefore hypothesised that the polymorphisms associated with increased cardiac adrenergic stimulation may increase the risk of progression to HF. The main findings of our study are that there was no difference in the frequency of ß1- and ß2-AR polymorphisms and their combinations among cases and controls.

There are few studies investigating the role of ß1-AR and ß2-AR polymorphisms as possible risk factors for HF. Tesson et al.,21 found no association between the Arg389Gly polymorphism and IDC in a case-control study comparing 426 patients and 395 controls. Other studies found no association between this polymorphism and the severity of heart failure,25 blood pressure and haemodynamics.31,32 However, Arg389Gly homozygosis is associated with a 3-fold increase in the cyclic AMP response to ß1-AR stimulation in vitro10 and the overexpression of this condition was recently found to recapitulate the end-stage dilated cardiomyopathy phenotype in a cardiac-targeted mouse model.33 Moreover, other clinical studies have shown significant correlations between the Arg389Gly polymorphism and haemodynamic parameters23 and the risk of hypertension.24 In our study, the patients with HF had a similar prevalence of Arg389 homozygosis compared to the normal subjects.

The substitution of Ser for Gly at position 49 in the gene encoding for ß1-AR has been associated with a worse prognosis in patients with HF.34 Gly49 homozygosis is uncommon, however, with a prevalence of 3% in one study34 and of 1.68% in another.35 Also our study population the prevalence was <5% and we did not therefore analyse its association with HF.

Many studies have assessed the role of ß2-AR polymorphisms (Arg16Gly, Gln27Glu) in cardiovascular diseases, mainly hypertension. Herrmann et al.,36 and Castellano et al.,37 found no association between these polymorphisms and the prevalence of hypertension, whereas Bray et al.,38 found that the frequency of both the Gly16 and the Glu27 allele was higher in hypertensives than in normotensives. Timmermann et al.39 found a significant association between the Arg16 allele and hypertension. There is also discordance regarding the functional significance of the ß2-AR polymorphism. An in vitro study showed that both Arg16 and Glu27 alleles were associated with resistance to agonist-promoted downregulation.17 By contrast, resistance to the ß2-mediated venodilatory response to isoproterenol was found in subjects homozygous for Gly16 and Glu27 or Gln27 but not in those homozygous for Arg16 in an in vivo study.18 This discrepancy may be explained by the dynamic regulation of ß2-AR, so that the subjects with the polymorphisms associated with increased sensitivity to ß2-AR downregulation may be "pre-desensitised" by their endogenous agonists and do not show it when further exposed to the agonist.40

A linkage disequilibrium is known to occur between the two ß2-AR polymorphisms19 and our study confirmed that all subjects homozygous for the Glu27 allele were also homozygous for the Gly16 allele. Therefore, only three haplotypes of the four homozygous combinations exist: Gly16Glu27, Gly16Gln27 and Arg16Gln27. However, we found no difference in their distribution between cases and controls.

We then hypothesised that some combinations of the three polymorphisms we studied may have a synergic effect on the incidence of HF. We hypothesised that homozygosis for Arg389, which is associated with enhanced sensitivity to ß1-AR stimulation,10 combined with the Gly16Gln27 haplotype, which is associated with increased ß2-AR downregulation,17 might predispose subjects to the development of HF. Although there was no significant difference in these combinations between cases and controls, a tendency to a higher risk of HF was found in subjects with the Arg389Gly16Gln27 genotype combination.

The relatively low number of subjects involved could be considered a major limitation of this study. However, this is the first study assessing the potential association between these ß-ARs polymorphisms and HF. In addition, our cases and controls were accurately matched with regards to age, ethnicity and area of residence. This is also shown by the lack of any deviation from the Hardy–Weinberg equilibrium in our study population. All the polymorphisms we analysed had an allele frequency >10% so that, according to our statistical calculations, the size of our study group allows the detection of significant differences between cases and controls with regards of SNP. However, a larger population is necessary to investigate the combinations of more polymorphisms (gene–gene interactions).

In conclusion, we did not find any role of ß1- and ß2-AR SNPas risk factors for HF. The analysis of haplotypes and allele combinations may show significant associations in larger study groups.


    Conflict of interest
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 Conflict of interest
 References
 
Authors have no financial associations that might pose a conflict of interest in connection with the submitted article to disclose. All sources of funding for the work have been acknowledged in a footnote on the title page. We have no other kinds of associations, such as consultancies, stock ownership or other equity interest or patent-licensing arrangements to disclose. We have no conflict of interest.


    Acknowledgments
 
This paper was partially supported by CARIPLO funds from the University of Brescia's Centro per lo studio del trattamento dello scompenso cardia co.


    References
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 Conflict of interest
 References
 

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