Effects of GRK5 and ADRB1 polymorphisms influence on systolic heart failure

G-protein receptor kinase 5 (GRK5) Gln41 > Leu and β1-adrenergic receptor (ADRB1) Arg389 > Gly polymorphisms presented the different distribution of genotype frequencies between Caucasian American and African American, and produced the difference in β-blocker treatment effect among them with systolic heart failure (SHF). This study sought to identify the distributed characteristics of these variant genotypes in Chinese population, and influence of GRK5 and ADRB1 polymorphisms on SHF morbidity and β-blocker treatment effect in patients with SHF. This study was based on cross-sectional survey data. 1794 and 1718 subjects’ ADRB1 and GRK5 gene sequencing (sanger method) data were achieved respectively. Blood samples collection, clinical laboratory detection, electrocardiogram and echocardiography examinations were performed. Medication usage was confirmed at in-hospital visits or the questionnaire by personal interview. GRK5 Leu41Leu genotype was not found in our Chinese population. In non-SHF population, allele frequencies of GRK5 Gln41 and Leu41 were 2782 (0.992) and 22 (0.008) (Hardy-Weinberg equilibrium test χ2 = 0.088, P = 0.767), and allele frequencies of ADRB1 Arg389 and Gly389 were 2127 (0.715) and 849 (0.285) (χ2 = 0.272, P = 0.602). In SHF patients, allele frequencies of Gln41 and Leu41 were 446 (0.991) and 4 (0.009) (χ2 = 0.018, P = 0.893), and allele frequencies of Arg389 and Gly389 were 331 (0.726) and 125 (0.274) (χ2 = 1.892, P = 0.169). Further in logistic regression model, these ADRB1 and GRK5 variants were not significantly independently associated with the risk of SHF morbidity. Those carrying genotype ADRB1 Gly389Gly did not reduce significantly the risk of SHF morbidity after β-blocker therapy. GRK5 Leu41Leu genotype was not found in our Chinese population, neither ADRB1 nor GRK5 variants presented independently associated with the risk of SHF morbidity, most ADRB1 and GRK5 polymorphisms did decrease significantly the risk of SHF morbidity after β-blocker therapy except for those carrying genotype ADRB1 Gly389Gly.


Introduction
Heart failure (HF) is an incurable syndrome arising from multiple causes that results in high morbidity rate, mortality rate and healthcare costs [1][2][3]. The management of HF is also complicated by disease heterogeneity in both inherited genetic cardiomyopathies [4] and the more common nonfamilial dilated and ischemic cardiomyopathies [5,6]. Thus efforts are underway to identify additional genetic markers that will indicate prognosis and guide management of patient care.
Pharmacogenomic interactions involving genetic variants of catecholamine receptors or their effectors (i.e., Gprotein receptor kinase 5 helps to terminate β-adrenergic receptor signaling by phosphorylating and uncoupling agonist-occupied receptors from their Gas signal transducers) [7] would be especially relevant given their important role in clinical disease management. Importantly, β adrenergic antagonism (β-blocker), which is a class I treatment indication for HF [8].
Previous pharmacogenomic studies have proposed that the β1-adrenergic receptor (ADRB1) Arg389 > Gly [9] and G-protein receptor kinase 5 (GRK5) Gln41 > Leu [10] polymorphisms, both of which are over-represented in African Americans, may play roles in determining individual clinical responses to β-blockade treatment with HF. Human studies have been inconsistent as to whether there are meaningful associations between this polymorphism and HF outcome [11][12][13].
Considering that ADRB1 and GRK5 polymorphisms were represented differently among ethnicities, and influenced the β-blocker treatment effect with systolic heart failure (SHF), thus our study was designed to identify whether GRK5 Gln41Leu and ADRB1 Arg389Gly polymorphisms would affect on SHF in Chinese population.

Study subjects
Our study was based on 2095 subjects' cross-sectional survey data (including seven patients with SHF) which has been reported previously [14,15], and other 253 patients with SHF derived from Shanghai East hospital, Pudong new district people's hospital, and Shanghai the 8 th people's hospital. After excluding some subjects with the missing partial clinical data and/or the blood samples. Finally, 1800 subjects' blood samples were delivered for ADRB1 and GRK5 polymorphisms sequencing. 1794 and 1718 subjects' ADRB1 and GRK5 gene sequencing data were achieved respectively. The study protocol was approved by the Institutional Review Board of the Shanghai East hospital affiliated with Tongji university (No, 2010020), and subjects gave written informed consent. Our study was performed to conform the declaration of Helsinki. Enrollment criteria for SHF were age of 30-75 years, a left ventricular ejection fraction (LVEF) less than 50% or two-dimensional echocardiography of mild or greater systolic dysfunction on visual assessment in multiple views [16] and class II-IV heart failure (new York Heart Association). Excluding criteria for SHF were the final stage of multiply organ dysfunction, severe anaemia, hyperthyreosis, psychosis, pregnancy and lactation.
Blood samples collection, clinical laboratory detection, electrocardiogram examination, echocardiography examination and relevant quality controls were described previously in our two studies [14,15]. Medication usage was confirmed at in-hospital visits or the questionnaire by personal interview.

Genotyping
Genomic deoxyribonucleic acid (DNA) for genotyping was isolated and extracted using KAPA Express Extract DNA kit (Kapa Biosystems Co, Boston). Homo sapiens adrenoceptor Beta 1 (ADRB1) single nucleoide polymorphism fragment (468 bp) 1165 C is replaced with G, which causes a nonconservative amino acid substitution of Gly for Arg at residue 389 [17], and Homo sapiens G protein-coupled receptor kinase 5 (GRK5) single nucleoide polymorphism fragment (399 bp) 122 A is replaced with T, which results in a nonconservative amino acid substitution of Leu for Gln at position 41 [10].
The PCR primers were designed using Primer 5.0 software (Premier Co, Canada), and PCR primer sequences were screened across the human genome using the National Center for Biotechnology Information Blast program to ensure their specificity for the gene of interest, (http://www.ncbi.nlm.nih.gov/tools/primer-blast/index. cgi?LINK_LOC=BlastHome website).

Statistical analysis
Data analysis was performed with SPSS (version 16.0 for Windows; SPSS Inc, Chicago) and stata 10.0 software (StataCorp, Texas). The normal distributions of numerical variables were described as mean ± SD. Categorical variables were expressed as percentage or proportion. One-way ANOVA and Chi-square test were used to assess significant differences of variables between genotype classes and between SHF group and non-SHF group. If the skew distributed data were expressed as median and quartile, Kruskal-Wallis H test and Mann-Whitney test were used for comparison of clinical variables between genotype classes and between SHF group and non-SHF group. The Hardy-Weinberg equilibrium was assessed using Chi-square test in each genotype group separately. Binary Logistic Regression was performed to assess the risk of SHF morbidity related to GRK5 and ADRB1 polymorphisms. A 2-tailed value of P < 0.05 was considered statistically significant.
Noticeably, we did not found significantly different of SHF morbidity between genotype classes (see Table 1). Further analysis indicated that age, heart rate, smoking, hypertension, diabetes mellitus, coronary heart disease, chronic renal disease, valvular heart disease, glycosylated hemoglobin and Hs-CRP were closely associated with SHF morbidity, however, ADRB1 and GRK5 variants did not present significantly independently associated with the risk of SHF morbidity (see Table 2). Furthermore GRK5 and ADRB1 polymorphisms did not present significantly difference between non-SHF group and SHF group (see Table 3). Importantly, the two functional βadrenergic receptor signaling polymorphisms in our population showed not significantly different of genotype frequency distribution and allele frequency distribution between non-SHF group and SHF group, thus this study suggested that GRK5 and ADRB1 polymorphisms did not induce the risk of SHF morbidity.
On the other hand, in 260 patients with SHF, there were 66.5 percent of patients without β-blocker treatment, and 20.4 percent of patients received metoprolol treatment, 10.0 percent of patients had bisoprolol, but carvedilol user only occupied 3.1 percent of SHF patients. It appeared to evidently different of beta-blockers usage between non-SHF group and SHF group (see Table 3). Further, we assessed whether GRK5 and ADRB1 polymorphisms were associated with the risk of SHF morbidity among β-blocker user, our findings showed that the naive patients carrying genotype ADRB1 Gly389Gly did not reduce significantly the risk of SHF morbidity after β-blocker therapy (Odd ratio (OR), 0.48; 95% confidence interval, 0.08-3.00; P = 0.415) except for other ADRB1 and GRK5 genotypes (see Figure 2).

Discussion
The findings of this study were that (1) GRK5 Leu41Leu genotype was not found in our Chinese population (see Figure 1); (2) Neither ADRB1 nor GRK5 variants presented independently associated with the risk of SHF morbidity; (3) most ADRB1 and GRK5 polymorphisms did reduce significantly the risk of SHF morbidity after β-blocker therapy except for those carrying genotype ADRB1 Gly389Gly. The β1-adrenergic receptor is the predominant subtype expressed in the human heart and plays an important role in the physiologic and pathophysiologic regulation of the cardiovascular system [18]. Genetic polymorphisms of the major cardiac β-blocker target, the β1-adrenergic receptor, and a kinase that terminates its signaling, GRK5, can significantly impact HF outcomes, and that adjusting for these gene variants abrogates the apparent ethnic differences in β-blocker treatment effect on SHF development [19]. It was reported that the minor allele frequency of GRK5 Leu41 was 32.6% in blacks and 0% in whites [20]. Another study indicated that among 600 UK and Dutch participants, the prevalence of the ADRB1 genotypes was Arg389Arg 51.3%, Arg389Gly 40.2%, Gly389Gly 8.5% [21]. In our non-SHF Chinese population, the genotype frequencies of Gln41Gln, Gln41Leu and Leu41Leu were 0.985, 0.015 and 0 respectively, allele frequencies of Gln41 and Leu41 were 0.992 and 0.008, which is accordant with those American whites [20], suggesting that the GRK5 homozygous mutation probability were very rare in our population. On the other hand, the genotype frequencies of ADRB1 Arg389Arg, Arg389Gly and Gly389Gly were 0.508, 0.413 and 0.079 in our Chinese population, allele frequencies of the minor ADRB1 Gly389 were 0.285, which was similar to the reported allele frequencies in normal Caucasian American subjects (Caucasians, 0.12 to 0.25 and African American, 0.23 to 0.38 respectively) [19] and European whites [21].
In experimental mouse models, overexpression of β adrenergic receptors (β ARs) [22] or their Gas G protein signaling transducer [23] causes cardiac dilation and failure. Conversely, genetic ablation of the β ARs and Gas downstream effector, adenylylcyclase, preserves myocardial function after physiological stress [24]. GRK5-Leu41 accelerated isoproterenol-promoted β ARs desensitization, suggesting that, like β-blockers, it might protect hearts from the effects of persistent β AR stimulation, that is, cardiac dilation, ventricular hypertrophy and heart failure [25]. Unfortunately, we did not found the GRK5 homozygous mutation (GRK5 Leu41Leu) in our population, which prevented us from further exploring its protection of SHF.
Lately a meta-analysis report included that 2642 cases and 3136 controls in 12 case-control population studies provided data on the association between Arg389Gly polymorphisms and susceptibility to idiopathic dilated cardiomyopathy, finally found that no significantly elevated idiopathic dilated cardiomyopathy risk was associated with Arg389Gly polymorphisms for all genetic models in all populations [26]. Similarly White HL et al also reported that ADRB1 Arg389Gly polymorphisms were not relevant to heart failure events among the patients in MERIT-HF research center of the UK and Holland [21]. The present study found that the allele frequencies of whether GRK5 Gln41 and Leu41 or ADRB1 Arg389 and Gly389 were similarly distributed between non-SHF population and SHF population, suggesting that GRK5 and ADRB1 Figure 2 GRK5 and ADRB1 polymorphisms influence on beta-blocker treatment with SHF. Note: CI denotes confidence interval. Advantage of beta-blocker indicated that the gene polymorphisms could decrease the risk of SHF morbidity after β-blocker therapy; Disadvantage of beta-blocker indicated that the gene polymorphisms could not reduce the risk of SHF morbidity after β-blocker therapy.
polymorphisms were not associated with SHF morbidity, further GRK5 and ADRB1 variants were not significantly independently associated with the risk of SHF morbidity (see Table 2). Thus our findings were consistent with the previous reports.
Given the reported associations between the β1 AR-Arg389 polymorphism and the response to β-blocker treatment with heart failure [9], the much higher norepinephrine affinity and adrenergic signal transduction capacity of Arg389 β1-AR may protect patients from excessive sympatholysis [27] that has been associated with increased mortality in β1 389-Gly carriers receiving bucindolol [28]. For other beta-blockers ADRB1 389Arg homozygotes may have greater improvements in LVEF compared with ADRB1 389Gly carriers [29,30], but no reduction in clinical endpoints has been demonstrated [12]. Latestly, bucindolol was associated with reductions in composite HF endpoints in those with the β1 389-Arg homozygous genotype [31]. Noticeably, the Arg389 variant of the β1adrenergic receptor was associated with a greater response to metoprolol than that of Gly389 in young, male Chinese subjects [32]. In our study, most ADRB1 and GRK5 polymorphisms did decrease the risk of SHF morbidity after β-blocker therapy, but those carrying genotype ADRB1 Gly389Gly had not likelihood of benefit from β-blocker therapy (see Figure 2).

Limitations
This was a cross-sectional data analysis, and it had better validate these findings prospectively. In addition, our data were derived from Han race in China, thus the findings were not generalized to other ethnicity in China.

Conclusion
GRK5 Leu41Leu genotype was not found in our Chinese population, neither ADRB1 nor GRK5 variants presented independently associated with the risk of SHF morbidity, most ADRB1 and GRK5 polymorphisms did reduce the risk of SHF morbidity after β-blocker therapy, but those carrying genotype ADRB1 Gly389Gly had not likelihood of benefit from β-blocker therapy.