Pharmacogenomics of Beta-Blockers and Statins: Possible Implications for Perioperative Cardiac Complications

EMERGING TECHNOLOGY REVIEW Gerard R. Manecke, Jr, MD Marco Ranucci, MD Section Editors

Pharmacogenomics of Beta-Blockers and Statins: Possible Implications for Perioperative Cardiac Complications Miklos D. Kertai, MD, PhD, Manuel Fontes, MD, and Mihai V. Podgoreanu, MD

P

ATIENTS UNDERGOING major noncardiac surgery can be at an increased risk for perioperative major adverse cardiac events because of the increasing prevalence of coronary artery disease (CAD) and congestive heart failure.1 A set of cardiac risk factors and noninvasive testing may help identify patients who can benefit from ␤-adrenergic-receptor blockers (␤-ADRBs) and, more recently, statins.2 These 2 classes of drugs have been shown independently to be effective in reducing perioperative and longterm nonfatal myocardial infarction and cardiac death.3-8 Furthermore, their combined use has been associated with a more pronounced reduction in perioperative cardiac complications across multiple levels of cardiac risk.9 However, not all patients are equally protected by ␤-ADRB and statin therapy,2 and, in some instances, patients treated with these agents may be at an increased risk for adverse drug reactions.10,11 Human genetic variations can be partially responsible for the clinical variation seen in response to ␤-ADRBs and statin therapy.12 Such genetic variants have been described in nonsurgical cardiovascular patients to affect the likelihood of experiencing adverse drug reactions as well as influencing therapeutic effect.13 This review summarizes clinical studies involving ␤-ADRB and statin therapy in patients with cardiovascular disease with an emphasis on genetic variation and its influence on the effect of these drugs at reducing perioperative complications. HUMAN GENETIC VARIATION AND PHARMACOGENETICS: A BRIEF OVERVIEW

The purpose of pharmacogenetics is to determine the genetic contribution to individual variation in response to pharmacotherapy, which functionally involves variation in both drug pharmacokinetics and pharmacodynamics. Many clinical characteristics are known to determine the optimal drug type and dosage, including age, sex, ethnicity, organ function, comorbidities, concomitant drug therapy, and drug interactions.12,14 Nevertheless, it has long been appreciated that variations in plasma drug concentrations are often attributable to genetic polymorphisms in drug-metabolizing enzymes. Several potential consequences of such a genetic variation in drug metabolism include adverse drug reactions, a decrease or lack of effectiveness, prolonged pharmacologic effect, drugdrug interactions, and metabolism by alternative pathways.13 Additional genetic variations affecting drug pharmacokinetics may influence the rates of absorption, distribution, metabolism, and excretion of drugs, whereas the most important functional conse-

quences of genetic variations affecting the pharmacodynamics of drugs are alterations in drug-receptor interactions.12-14 A single nucleotide polymorphism (SNP) is a variation in the DNA sequence that occurs when 1 nucleotide (ie, adenine, thymine, cytosine, or guanine) in the genome sequence is altered and is present with a frequency of at least 1% in a population. One SNP occurs at every 100 to 300 nucleotide base pairs (bps) in the 3 billion nucleotide human genome and can be located both in the coding or noncoding regions of a gene as well as in the intergenic regions. SNPs that occur in the coding region of the gene are classified as nonsynonymous if the change in the nucleotide bp results in an amino acid substitution or synonymous if the bp does not affect the amino acid sequence. Nonsynonymous SNPs potentially can change protein structure, stability, and/or structure as well as change substrate affinities and function or alter regulatory DNA sequences that modulate protein expression. SNPs that occur in the noncoding regions of a gene can still be functionally important by affecting gene expression, transcription factor binding, splicing, messenger RNA degradation, or the sequence of noncoding RNA. A set of nearby SNPs on a single chromosome that is transmitted together is referred to as a “haplotype.” Haplotype analysis has important roles in disease-related gene discovery. Other more complex types of human genomic variations are the so-called structural genetic variants, such as short-sequence repeats (microsatellites), the insertion/deletion of 1 or more nucleotides, inversions, and copy-number variants, which are large segments of DNA that differ in number of copies.13 ␤-ADRBs

␤-ADRBs frequently are prescribed perioperatively for blood pressure and heart rate control.2 The beneficial effect of

From the Department of Anesthesiology, Duke Perioperative Genomics Program, Duke University Medical Center, Durham, NC. Address reprint requests to Miklos D. Kertai, MD, PhD, Department of Anesthesiology, Duke University Medical Center, 2301 Erwin Road, 5693 HAFS Building, DUMC 3094, Durham, NC 27710. E-mail: [email protected] © 2012 Elsevier Inc. All rights reserved. 1053-0770/2606-0024$36.00/0 http://dx.doi.org/10.1053/j.jvca.2012.06.025 Key words: perioperative cardiac complication, ␤-blockers, statins, pharmacogenomics

Journal of Cardiothoracic and Vascular Anesthesia, Vol 26, No 6 (December), 2012: pp 1101-1114

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KERTAI, FONTES, AND PODGOREANU Table 1. Pharmacokinetics of ␤-ADRBs: Common and Functionally Significant CYP2D6 Polymorphisms Minor Allele Frequencies

CYP2D6 Allele/Haplotype

Effect on Enzyme Metabolism

Mutation

Whites

Blacks

3 4 5 6 9 10 17 1 2

Inactive* Inactive Inactive Inactive Partially active Partially active Partially active Normal Normal Gene duplication UM†

2549A ⬎ del Defective splicing Gene deletion 170T ⬎ del 2613-2615 delAGA 100C ⬎ T 1023C ⬎ T, 2850C ⬎ T None (wild-type) ⫺1584C ⬎ G, 1661G ⬎ C, 2850C ⬎ T, 4180G ⬎ C

0.01-0.04 0.12-0.21 0.02-0.07 0.01 0-0.02 0.01-0.02 0 0.33-0.36 0.22-0.33

0 0.06-0.08 0.06-0.07 0 0 0.03-0.08 0.15-0.23 0.29-0.35 0.18-0.27

0.02

0.01-0.05

UM

Abbreviation: UM, ultrarapid metabolizer. Adapted with permission from Nagele122 and modified using data from http://www.cypalleles.ki.se/cyp2d6.htm.20 *Poor metabolizers: a complete lack of CYP2D6 enzyme activity. Treatment with standard recommended doses of ␤-adrenergic-receptor blockers metabolized through CYP2D6 may produce higher drug plasma concentrations with an increased risk for side effects and drug toxicity.20 †Ultrarapid drug metabolism: excessively high CYP2D6 enzyme activity. Ultrarapid metabolizers may require higher-than-standard recommended doses of ␤-adrenergic-receptor blockers metabolized via CYP2D6 to achieve therapeutic effect.20

␤-ADRBs consists of reducing heart rate and contractility, resulting in a lengthening of the diastolic filling period, reduced shear stress, and an overall lowering of myocardial oxygen demand.15 Their therapy has been recommended for patients with known ischemic heart disease scheduled for high-risk noncardiac surgery to reduce the risk of perioperative myocardial infarction.2 However, the benefits of ␤-ADRB therapy recently have been called into question by reports of side effects, such as hypotension and bradycardia, which have been associated with significantly increased incidences of perioperative death and nonfatal stroke.16 Moreover, after accounting for demographic and clinical characteristics, the efficacy and toxicity of ␤-ADRBs in the treatment of hypertension,17 CAD,18 and heart failure19 may vary significantly. A number of genetic variants with a substantial influence on the efficacy and toxicity of ␤-ADRBs have been characterized, including inherited differences in the metabolism and disposition of ␤-ADRBs, as well as genetic polymorphisms in the therapeutic target of ␤-blockers (␤-adrenergic receptors and their signaling pathways). PHARMACOKINETICS OF ␤-ADRBs

One of the most extensively studied and characterized examples of pharmacogenetic variation is the cytochrome P450 2D6 isoenzyme, or CYP2D6, which is involved in the hepatic elimination of several drugs, including analgesics, antiarrhythmics, antidepressants, and most ␤-ADRBs. The CYP2D6 is highly polymorphic; at least 80 functional variants having been identified.20 Based on the presence of polymorphisms and the number of copies of functional alleles, patients can be stratified as ultrarapid metabolizers, extensive metabolizers (EMs, the normal phenotype), intermediate metabolizers, and poor metabolizers (PMs).21 The prevalence of different CYP2D6 enzyme polymorphisms varies by ethnicity (Table 1).22,23 Several of the lipophilic ␤-ADRBs (ie, metoprolol, propranolol, carvedilol, labetalol and timolol but not atenolol) are metabolized partially by the CYP2D6 isoenzyme. Metoprolol is

the most dependent on this enzyme, with 70% to 80% of its metabolism directed through this pathway.24 According to clinical studies, subjects who were CYP2D6 PMs had 3- to 10-fold higher plasma concentrations of metoprolol compared with subjects who were EMs with normal CYP2D6 activity.25,26 Furthermore, the elimination half-life of metoprolol was found to be 7.5 hours in PMs versus 2.8 hours in EMs.25 In the study subjects who were PMs, these observed differences in metoprolol plasma concentration and elimination half-life translated to a significantly more pronounced and prolonged decrease in heart rate during exercise.25 Subsequent studies replicated these observations. Indeed, patients who are CYP2D6 PMs are at a significantly higher risk for bradycardia27,28 and hypotension27,28 and for developing adverse effects29 during metoprolol treatment. In the setting of acute myocardial infarction, patients who were ultrarapid metabolizers and were started on metoprolol had lower trough metoprolol plasma concentrations, higher mean heart rates, and a higher incidence of ventricular rhythm disturbance compared to patients with other CYP2D6 genotypes.30 The evidence from these studies indicates that CYP2D6 polymorphisms can significantly influence ␤-ADRB pharmacokinetics, particularly in metoprolol. However, the clinical use of these pharmacokinetic genetic variants remains limited in terms of managing long-term risk of cardiovascular complications because doses of ␤-ADRBs can be titrated slowly.31 By contrast, studies on how genetic polymorphisms of CYP2D6 influence the efficacy of ␤-ADRBs in preventing perioperative cardiovascular complications are completely lacking. Nevertheless, in a recent meta-analysis, Badgett et al10 formulated an intriguing hypothesis for why some clinical trials failed to show a beneficial effect of perioperative ␤-ADRBs in preventing cardiac complications. They observed increased mortality in patients who received perioperative ␤-ADRBs with CYP2D6 metabolism and a short titration period. When the investigators tested for an interaction between the duration of the titration period and CYP2D6 metabolism, they found that

PHARMACOGENOMICS OF ␤-BLOCKERS AND STATINS

1103

Fig 1. Results of a meta-regression of odds ratios of mortality from ␤-ADRBs based on the length of the titration period and CYP2D6 metabolism. The risk of mortality from perioperative ␤-ADRBs was confined to studies that combined short titration period with CYP2D6 metabolism (Ln [odds ratio] of 0 indicates odds ratio ⴝ 1. Values of Ln <0 indicate benefit from ␤-ADRBs). BBSA, ␤-Blocker in Spinal Anesthesia; DECREASE I, Dutch Echocardiographic Cardiac Risk Evaluation Applying Stress Echocardiography I; DECREASE IV, Dutch Echocardiographic Cardiac Risk Evaluation Applying Stress Echocardiography IV; DIPOM, Diabetes Postoperative Mortality and Morbidity; MSPI, Multicenter Study of Perioperative Ischemia Research Group; POISE, Perioperative Ischemic Evaluation). (Adapted with permission from Badgett.10)

the highest mortality was observed in trials that used ␤-ADRBs with CYP2D6 metabolism and a short titration period (Fig 1).10 Importantly, there was no interaction observed between the route of drug metabolism and the risk for stroke in patients receiving ␤-ADRBs. These findings support the notion that the efficacy of ␤-ADRBs metabolized via the CYP2D6 enzyme is affected by relevant genetic polymorphisms. Therefore, when there is an indication for preoperative initiation of ␤-ADRBs, drugs that do not require a long period of titration and reliance on hepatic elimination, such as atenolol, should be considered.10 PHARMACODYNAMICS OF ␤-ADRBs

Genetic Polymorphisms of the ␤-ADRBs: ␤1Adrenergic Receptor The ␤1-adrenergic receptor is encoded by an intronless gene located on chromosome 10q24-26.32 Predominantly expressed in the myocardium (Fig 2), ␤1-adrenergic receptor stimulation is associated with positive inotropic and chronotropic effects in the heart.19 Several polymorphisms involving the ␤1-adrenergic receptor have been described,33-35 but 2 of these are prevalent and can significantly influence the efficacy and, possibly, the safety of ␤1-ADRBs. One of these polymorphisms is located in the amino terminus where an adenine-to-guanine exchange occurs at 145 bp, resulting in an amino acid substitution of serine (Ser) by glycine (Gly) at residue 49 (Ser49Gly) (Fig 2). The allele frequency of Gly49 varies between whites (about

15%-22%) and blacks (about 13%-22%).14 According to in vitro studies, amino acid 49 polymorphisms of ␤1-adrenergic receptor gene affect agonist-promoted trafficking, and the Gly49 receptor is characterized by enhanced agonist-promoted downregulation.35 Another SNP results from a guanine-to-cytosine exchange at 1,165 bp causing a nonconservative amino acid substitution of arginine (Arg) by glycine (Gly) at residue 389 (Arg389Gly) (Fig 2).33 The allele frequency of the Gly389 also varies between whites (about 27%) and blacks (about 42%).14 In vitro studies reported that the substitution markedly alters the G protein coupling of the ␤1-adrenergic receptor, with the Arg389 receptor having nearly 2-fold greater basal and 3-fold greater agonist-mediated adenyl cyclase activities.33 Human studies confirmed these findings by showing a significantly higher resting heart rate and diastolic blood pressure among subjects homozygous for the Arg389 genotype compared with those with other codon 389 genotypes. The codon 49 and 389 polymorphisms are in linkage disequilibrium, and studies suggest that haplotypes must be considered in determining the in vivo functional role of these polymorphisms in this important drug target.36 The presence of the Arg389 form of the receptor is associated with greater ␤1-adrenergic receptor activation,33,37 whereas the Ser49 form of the receptor has been most consistently associated with resistance to receptor downregulation.35,38 Therefore, the Ser49-Arg389 haplotype is expected to be the most responsive

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•

KERTAI, FONTES, AND PODGOREANU

•

Increased expression: increased heart rate and contractility; altered intraoperative hemodynamics in cardiac surgery

•

Pre-synaptic sympathetic nerve terminal

Gαs

GRK5

Gαs

•

Greater agonistpromoted receptor downregula
on

β1-AR Ser49Gly

Resistant to receptor downregula
on: reduced longterm mortality

•

Greater agonistpromoted receptor downregula
on: intraopera
ve hypotension

β2-AR Gln27Glu

β2-AR Arg16Gly

β3-AR Trp64Arg

-

+

β γ

Decreased agonistpromoted coupling: associated cardiovascular risk factors including obesity, diabetes, and hypertension

NH2

αi

Nepi p

γ Extracellular α2C-AR

β2 R

β

Epi

R

Gααs

+

Cell Membrane

β3-AR

β2-AR

COOH βγ

Intracellular

Gααss β γ

β2-AR Thr164Ile

Gαi β γ

Cardiomyocyte C

AC

-

+

•

Loss of function: more aggressive coronary artery disease

AC

cAMP cA AMP

α2C-AR Del322-325 • •

cAMP GRK5

Loss of function Sustained norepinephrine release: progressive heart failure

β1-AR Arg389Gly •

•

Decreased coupling: attenuated β-adrenergic cascade Possible synergistic effect when β-adrenergic receptor blockers used

41L Gln41Leu •

modifies β-adrenergic receptor signaling pathway: partial β-receptor blockade

Fig 2. Presynaptic nerve terminal, cardiac adrenergic receptors, and adrenergic-receptor signaling in the heart. Localization of the common polymorphisms of the adrenergic receptors with their functional consequences. A coupling of ␤2-adrenergic receptor to stimulatory G protein (Gs) and inhibitory G protein (Gi) is shown in the cardiomyocytes but not in the presynaptic nerve terminal. AC, adenyl cyclase; AR, adrenergic receptor; cAMP, cyclic adenosine monophosphate; Epi, epinephrine; GRK5, G protein-coupled-receptor kinase; Nepi, norepinephrine. (Adapted from Johnson and Liggett by permission from Macmillan Publishers Ltd.18 Copyright 2011.)

to activation by catecholamines, and, consequently, a greater response to ␤-ADRB also is expected.39 These genetic variations in the ␤1-adrenergic receptor gene could be responsible for interindividual differences in response to therapeutic ␤-adrenergic-receptor agonists and antagonists in cardiovascular and other diseases including in the perioperative period. This is suggested by previous epidemiologic studies reporting associations of ␤1-adrenergic-receptor polymorphisms with a variety of intermediate cardiovascular phenotypes, cardiovascular risk, and ␤-ADRB responses in hypertension and heart failure.17,39-42 In the Swiss ␤-blocker in Spinal Anesthesia Study,43 ␤1adrenergic receptor polymorphism determined the postoperative long-term survival of patients who underwent noncardiac surgery with spinal block anesthesia. Patients with the Arg389 genotype had significantly lower incidences of cardiovascular morbidity and mortality compared with patients with the Gly389 genotype. However, the selective use of ␤1-ADRB did not affect cardiovascular outcome in patients with the Arg389 or the Gly389 genotype compared with placebo.43

In a more recent study, Parvez et al44 studied the impact of ␤1-adrenergic-receptor polymorphisms on ventricular rate control therapy in patients with atrial fibrillation. They found that carriers of the Gly389 genotype were more likely to respond to rate-control therapy than patients with the Arg389 genotype (60% v 51%, p ⫽ 0.04). In fact, patients with the Gly389 variant who were responders required the lowest doses of rate-control medications, including ␤-ADRBs and calcium channel blockers. The authors postulated that the Gly389 genotype (resulting in a loss-offunction ␤1-adrenergic-receptor variant with attenuated ␤-adrenergic signaling cascade) and the addition of ␤-ADRBs could have synergistic effects, which explains the observed findings of adequate response to rate-control therapy in patients with atrial fibrillation.44 Overall, the results from these studies suggest that genetic heterogeneity of the ␤1-adrenergic receptor translates to differences in clinical outcome. These findings also imply that, in future trials of perioperative ␤-ADRB therapy, investigators should consider genetic stratification of the study

PHARMACOGENOMICS OF ␤-BLOCKERS AND STATINS

population to maximize the efficacy of ␤-ADRB therapy and limit toxicity. Genetic Polymorphisms of the ␤-Adrenergic Receptors: ␤2-Adrenergic Receptor The gene encoding the ␤2-adrenergic receptor is located on chromosome 5q31-q32.45 The ␤2-adrenergic receptor has been found in all cell types, including heart and blood vessels (Fig 2). In the vascular system, ␤2-adrenergic-receptor stimulation mediates smooth muscle cell relaxation, whereas in cardiac myocytes it is associated with increased contractility, heart rate, and antiapoptotic effects.19 Several important genetic polymorphisms of the ␤2-adrenergic-receptor gene have been described, but 3 of these polymorphisms have been particularly well studied because of their effects on asthma and cardiovascular function, morbidity, and mortality.19 These 3 important nonsynonymous polymorphisms are at amino acid positions 16 (arginine to glycine [Arg16Gly]), 27 (glutamine to glutamic acid [Gln27Glu]), and 164 (threonine to isoleucine [Thr164Ile]). The Gly16 and Glu27 polymorphisms are common, with minor allele frequencies between 40% and 50% in whites and blacks.19 In contrast, the Ile164 variant is rare, with minor allele frequency of 1% in whites and ⬍2% in blacks.19 The polymorphisms at amino acid position 16 and 27 are in the extracellular amino terminus of the ␤2-adrenergic receptor and have an enhanced effect on agonist-promoted receptor downregulation.46 The polymorphism at amino acid position 164 is in the fourth transmembrane domain of the ␤2-adrenergic receptor, with the variant type of the polymorphism associated with defective receptor coupling to the stimulatory G protein, impaired agonist-promoted sequestration, and lower agonist binding affinity.47 Results of studies on the effect of the Gly16 variant on cardiovascular outcomes are inconsistent. Case-control studies in patients with CAD found no association between the Gly16 variant and cardiovascular morbidity and mortality.48,49 There was also no interaction observed between ␤-ADRB use and this gene variation on the risk of myocardial infarction and ischemic stroke.49 In contrast, in the Swiss ␤-blocker in Spinal Anesthesia Study,43 it was observed that the Gly16 variant was more associated frequently with intraoperative hypotension compared with the Arg16 variant, but this effect was independent of ␤-ADRB use. In patients with idiopathic dilated cardiomyopathy, the Gln27 allele of the ␤2-adrenergic-receptor gene is associated with a lower risk for heart failure, cardiac transplantation, and death from heart failure (hazard ratio ⫽ 0.15; 95% confidence interval, 0.05-0.42; p ⬍ 0.001) (Fig 2).50 In a hypertensive population, the Gln27 variant in the presence of the Gly16 variant is associated with a higher systolic blood pressure, but these variants showed no association with an increased risk for myocardial infarction.51 In patients with acute coronary syndrome, the association between the Gln27 variant and higher long-term mortality was dependent on whether the patient was discharged on ␤-ADRB therapy.52 Among patients treated with ␤-ADRBs, the mortality rate was 16% in patients who were homozygous for the Gln27 variant compared with 11% in heterozygous patients and 6% in

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patients who were homozygous for the Glu27 variant. Among patients treated with ␤-ADRBs, patients with a ␤2-adrenergicreceptor polymorphism at amino acid position 16 had similar mortality rates depending on whether the patients were homozygous for Arg16 (20%), heterozygous (10%), or homozygous for the Gly16 variant (10%). When haplotypes of the Arg16Gly and Gln27Glu variants were considered, the risk for long-term mortality was highest (20%) among patients homozygous for both Arg16 and Gln27 compared with the risk for mortality (6%-11%) in other haplotypes. However, no association was identified between Arg16Gly or the Gln27Glu variants and higher long-term mortality among patients not discharged on ␤-ADRB therapy.52 The third, least frequently nonsynonymous polymorphism of the ␤2-adrenergic receptor, is the Thr164Ile variant (Fig 2). This variant is associated with reduced G-protein coupling and decreased ligand affinity, which cause impaired vasodilation and increased peripheral vascular resistance.53 Accordingly, subjects heterozygous or homozygous for the Ile164 variant may have a higher frequency of hypertension and, subsequently, can be at higher risk for cardiovascular disease.54 In a recent, large-scale prospective cohort study, Thomsen et al54 found that, in women, Ile164 heterozygosity and homozygosity were associated with the severity of blood pressure elevation, the frequency of hypertension, and the increased risk of cardiovascular disease. A prospective study in patients with heart failure found that patients with the Ile164 variant were at a higher risk for death or cardiac transplantation compared with patients with the Thr164 variant.55 In a more recent study, the Ile164 variant was not associated with a higher risk for death in patients with heart failure; however, the results of a multivariate analysis revealed that ␤-ADRB therapy negatively impacted survival in the Ile164 group.56 The effects of the Ile164 variant on the development and complications of CAD also were studied in patients who underwent percutaneous coronary artery intervention.57 Patients heterozygous for the Ile164 variant had an earlier onset of CAD and a higher incidence of multivessel disease compared with patients homozygous for the Thr164 variant. At follow-up, patients carrying the Ile164 variant compared with patients with the Thr164 variant had a higher incidence of myocardial infarction (17.5% v 4.5%, p ⫽ 0.001), new percutaneous coronary intervention (37.5% v 13.1%, p ⬍ 0.0001), and higher cardiac mortality (10% v 3.1%, p ⫽ 0.04). Furthermore, in a multivariate analysis, patients with the Ile164 variant were also 3.7 times more likely to have cardiac death and 4.1 times more likely to have major adverse cardiac events. Finally, when the results of the study were validated in a group of patients with peripheral vascular disease, patients with the Ile164 variant had a higher incidence of myocardial infarction (54.5% v 25.2%, p ⫽ 0.035) and major adverse cardiac events (63.6% v 30.9%, p ⫽ 0.03) compared with patients homozygous for the Thr164 variant.57 Thus, in patients undergoing percutaneous coronary intervention for CAD, the presence of the Ile164 variant appears to be associated with a more aggressive type of coronary atherosclerosis. Of note, in relation to cardiovascular complications, this study failed to evaluate the effect of ␤-ADRB therapy on cardiovascular complications in patients with the Ile164 variant.

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In summary, studies have indicated that ␤2-adrenergic-receptor polymorphisms are clinically important in modulating the risk for cardiovascular morbidity and mortality. However, conflicting results have been reported regarding the role of ␤-adrenergic-receptor blocker use in the context of ␤2-adrenergic-receptor polymorphisms for the prevention and treatment of cardiovascular disease. Genetic Polymorphisms of the ␤-ADRBs: ␤3-Adrenergic Receptor The gene encoding the ␤3-adrenergic receptor is located on chromosome 8p11-8p12.58 The ␤3-adrenergic receptor has been found in several tissues, including the heart and blood vessels. In the vascular system, ␤3-adrenergic-receptor stimulation in both endothelial and smooth muscle cells leads to vasodilation. Stimulation of the ␤3-adrenergic receptor in the cardiomyocytes is associated with a negative inotropic effect, a positive lusitropic effect, and antihypertrophic properties.59 One nonsynonymous polymorphism of the ␤3-adrenergicreceptor gene has been described. It is characterized by a substitution of arginine for tryptophan at amino acid position 64 (Trp64Arg) and is associated with cardiovascular risk factors such as obesity, diabetes mellitus, and hypertension.60 This polymorphism is located either in the first transmembranespanning domain or in the first of the 3 intracellular loops of the receptor (Fig 2).19 The minor allele frequency of the Arg64 variant varies with race; it is less prevalent in whites (8%), blacks (12%), and Mexican Americans (13%) compared with Pima Indians (31%).61 In vitro studies on the pharmacologic effect of the Arg64 variant report conflicting results. Candelore et al62 found no difference between the Arg64 variant and the Trp64 variant in agonist-binding characteristics or in intracellular cyclic adenosine monophosphate accumulation. In contrast, Piétri-Rouxel et al63 observed that the Arg64 variant is associated with attenuated agonist-promoted coupling with reduced intracellular cyclic adenosine monophosphate accumulation compared with the Trp64 variant. A case-control study and a simultaneous meta-analysis in patients at risk for cardiovascular disease failed to show evidence for a role of the Arg64 variant in acute myocardial infarction and CAD.64 There is a lack of data on the effect of the Arg64 variant on the response to ␤-adrenergic-blocker therapy. However, a recent study reported that nebivolol, a selective ␤1-adrenergicreceptor antagonist, has ␤3-adrenergic-receptor agonist properties, which may explain the beneficial effect of nebivolol on survival in patients with chronic heart failure.65 Nebivolol could lead to myocardial protection by reducing intracellular calcium overload, improving diastolic function through increased nitric oxide production, and reducing afterload through vasodilation.66 Genetic Polymorphisms of the Intracellular Signaling System: G Protein–Coupled-Receptor Kinase 5 The role of G protein– coupled-receptor kinase (GRK) is to specifically recognize and phosphorylate agonist-activated G protein– coupled receptors, such as the ␤-adrenergic receptors. This initiates deactivation of these receptors, which is a major mechanism for ␤-adrenergic-receptor desensitization.67 Of the

KERTAI, FONTES, AND PODGOREANU

7 isoforms in the GRK family present in humans, GRK5 and GRK2 are highly expressed in the myocardium.67 The GRK5 gene is located on chromosome 10q26.11-26.11. A recent study found that a genetic polymorphism of GRK5 can modify signaling through the ␤-adrenergic receptors and can, thus, impart a pharmacogenomic effect on ␤-ADRB responsiveness.68 This polymorphism is more prevalent in blacks (23%-35%) than in whites (1%-2.4%).68 In a series of in vitro and animal studies, Liggett et al68 found that the GRK5-leucin (Leu41) variant is markedly more effective than the GRK5-glutamine (Gln41) variant in blunting the effects of ␤-adrenergic-receptor agonists via enhanced receptor desensitization (Fig 2). In fact, GRK5-Leu41 showed a protective effect much like ␤-ADRBs. In the same study, the investigators also examined the effect of this polymorphism in 2 cohorts of black patients with heart failure. They found that the GRK5-Leu41 variant protected against early death and cardiac transplantation. These findings were corroborated in a prospective, large-scale study of heart failure patients.69 Cresci et al69 observed that the GRK5-Leu41 variant is associated with improved long-term survival in blacks. In summary, these findings indicate that the GRK5-Leu41 variant modifies the ␤-adrenergic, receptor signaling pathway in a way similar to the partial ␤-adrenergic-receptor antagonism by ␤-ADRBs.68 However, it is not known yet how the effect of this genetic variation may influence the patient response to ␤-ADRBs in the perioperative period. Genetic Polymorphisms of the Intracellular Signaling System: Stimulatory G Protein–Alpha-Subunit The stimulatory G protein–␣-subunit (G␣s) has an important role in maintaining and augmenting left ventricular function and hypertrophy.70 The gene encoding G␣s is located on chromosomes 20q13 and 32. Genetic polymorphisms in the regulatory regions of the Guanine Nucleotide Binding Protein, Alpha Stimulating (GNAS) gene influence the function and expression of G␣s and may impact long-term survival.71 The increased expression of G␣s has been associated with increased heart rate and cardiac contractility,70 which also may influence the long-term prognosis of patients with CAD undergoing cardiac surgery (Fig 2).71 Recently, Frey et al72 identified a certain haplotype in the GNAS gene associated with G␣s expression that altered intraoperative hemodynamics in patients undergoing coronary artery bypass surgery. In study patients who were chronic ␤-ADRB users, cardiac performance was dependent on G␣s expression. Under ␤-ADRBs, patients with a homozygous haplotype associated with increased G␣s expression had higher stroke volume and cardiac index and lower N-terminal pro B-type natriuretic peptide concentrations than patients with a heterozygous or with a negative haplotype.72 Genetic Polymorphisms Associated With ␤-ADRB Efficacy: A2C-Adrenergic Receptor The ␣2C-adrenergic-receptor gene is located on chromosomes 4p16 and 3. The ␣2C-adrenergic receptor plays a critical role in regulating norepinephrine release from presynaptic nerve terminals in the heart. Stimulation of the ␣2C-adrenergic

PHARMACOGENOMICS OF ␤-BLOCKERS AND STATINS

receptor by epinephrine or norepinephrine results in decreased norepinephrine release from the presynaptic nerve terminals (Fig 2).19 An insertion/deletion polymorphism in the ␣2C-adrenergic receptor influences the therapeutic efficacy of ␤-ADRBs in cardiovascular disease.18 This polymorphism is localized to the third intracellular loop of the receptor and is characterized by a 12-nucleotide in-frame deletion resulting in a loss of 4-amino acids (glycine-alanine-glycine-proline [␣2C-Del322-325]).19 The population frequency of the ␣2C-Del322-325 variant is approximately 40% in blacks and approximately 4% in whites.18 The ␣2C-Del322-325 variant results in a loss of function, which is associated with decreased coupling of the receptor to the inhibitory G protein.73 Small et al74 hypothesized that sustained norepinephrine release is associated with this deletion variant, which may cause progressive heart failure. Clinical studies in heart failure patients reported that patients with the ␣2C-Del322-325 variant had worse clinical status and a lower left ventricular ejection fraction than patients without this deletion.75 Patients with the ␣2C-Del322-325 variant (heterozygous or homozygous) experienced a much greater sympatholytic effect when treated with bucindolol, a ␤-ADRB with a sympatholytic effect, compared with patients with the normal variant of the receptor.76 In a different study of heart failure patients treated with metoprolol, a synergistic effect between the Arg389Gly polymorphism and the ␣2C-Del322-325 polymorphism was observed. This resulted in the greatest improvement in ejection fraction compared with patients with the other variants of the ␤1-adrenergic and ␣2C-adrenergic receptors.77 However, in patients with cardiac risk factors who are undergoing surgery, it remains unknown how the effect of the ␣2C-Del322-325 polymorphism may influence clinical therapeutic responses to ␤-ADRBs, with different pharmacologic properties. PHARMACOGENETICS OF STATIN THERAPY

3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase Inhibitors (Statins) Statins have become one of the most frequently prescribed cardiovascular medications in patients with or at risk for ischemic heart disease. Guidelines also indicate statin use for secondary prevention in patients with noncoronary atherosclerotic disease.78 Statins have beneficial effects on atherosclerosis and vascular properties that are not attributed to their lipidlowering effect.79 These so-called pleiotropic effects may attenuate coronary artery plaque inflammation and also may have antithrombogenic, antiproliferative, and leukocyte adhesion inhibiting properties.80 All of these pleiotropic effects of statins may lead to stabilization of unstable coronary plaques, thereby reducing myocardial ischemia and myocardial infarction in the perioperative period.81 The beneficial effects of perioperative statin use have been shown in clinical trials and observational studies.1 Although the findings of these studies indicated that statins are efficacious in preventing perioperative and long-term cardiac morbidity and mortality, not all patients were protected by statin use, regardless of their risk level for cardiac complications.1 Recently, considerable interindividual differences in responsiveness to

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statins have been observed in patients with ischemic heart disease, which, aside from nongenetic factors, such as age, concomitant medication use, and comorbidities, have been linked to genetic variability.82 PHARMACOKINETICS OF STATINS: GENETIC POLYMORPHISMS ASSOCIATED WITH THE PLEIOTROPIC EFFECTS

Genetic Modifiers of Statin Pharmacokinetics: Cytochrome P450 Enzymes The cytochrome P450 (CYP) enzyme system plays a prominent role in the pharmacokinetics of most statins (Fig 3). The CYP2C9 isoenzyme is responsible for metabolizing fluvastatin and pravastatin, and the CYP3A4 isoenzyme is responsible for metabolizing atorvastatin, lovastatin, and simvastatin. However, the metabolism of some statins, such as pivastatin and rosuvastatin, occurs independent of the P450 (CYP) enzyme system.82 Several SNPs of the P450 (CYP) enzyme system are involved in the pharmacokinetics of statins.83 However, studies have reported conflicting findings regarding the role of these genetic polymorphisms in influencing the plasma concentration of statins and, thus, in determining cholesterol-lowering efficacy and patient tolerance to statin therapy.83 Furthermore, these studies have found that these genetic polymorphisms do not significantly influence the risk of cardiovascular complications by changing the pleiotropic effects of statins.83 Genetic Modifiers of Statin Pharmacokinetics: P-Glycoprotein Transporter A genetic variation in the ABCB1 gene that encodes for the P-glycoprotein transporter has been implicated in the variability of responsiveness to statins.84 This gene is located on chromosomes 7q21 and 12.85 The P-glycoprotein transporter, also known as the ABCB1 transporter, belongs to the adenosine triphosphate– binding cassette (ABC) superfamily. It is an efflux transporter responsible for actively transporting substrates, such as statins outside of the liver cells (Fig 3).86 Many SNPs of the ABCB1 gene have been identified, but 3 common polymorphisms (C1236T [glycine412glycine], G2677T/A [serine89alanine/thyrosine], and C3435T [isoleu cine1145isoleucine]) are in linkage disequilibrium and jointly contribute to the common haplotypes (1236C-2677G3435C [CGC] and 1236T-2677T-3435T [TTT]) implicated in the variability of ABCB1 transporter functioning.82 ABCB1-related alleles are the most common variants, with an overall frequency of 43% in Americans with European ancestry and 69% in blacks.87 The C3435T variant affects the timing of cotranslational folding of the ABCB1 transporter,88 which can result in altered statin specificity.89 In a recent study, subjects carrying the ABCB1 3435T genotype and/or ABCB1 TTT haplotype had lower ABCB1 transporter activity. This was associated with enhanced intestinal absorption and impaired hepatobiliary excretion of statins (eg, simvastatin, atorvastatin, and lovastatin).90 Several studies reported that subjects with the variant type of the ABCB1 transporter had a better lipid response.84,91,92 In contrast, in subjects carrying the ABCB1 3435T genotype and/or ABCB1 TTT

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KERTAI, FONTES, AND PODGOREANU

Fig 3. A generalized view of the pharmacokinetics of statins and the set of all genes reported to influence statin transport and metabolism. Statins are dosed orally and enter the systemic circulation through intestinal cells both passively and by active transport via the ABC and solute carrier (SLC) gene family transporters. The metabolism of statins is catalyzed by the cytochrome P450 (CYP) and glycosyltransferase (UGT) gene family enzymes and takes place in the liver. The main pathway of elimination is ABC transporter–mediated biliary excretion. All of these genes vary in their affinity for different statins and statin metabolites. For additional information, please visit the Pharmacogenetics and Pharmacogenomics Knowledge Database (PharmGKB, http://www.pharmgkb.org/pathway/PA145011108?previousQueryⴝstatins#). Colors are as follows: pink, drug or metabolite; blue, transporter protein; purple, metabolizing enzyme. (Reprinted from McDonagh with permission from PharmGKB and Stanford University. Copyright PharmGKB.121)

PHARMACOGENOMICS OF ␤-BLOCKERS AND STATINS

haplotype, the higher hepatic exposure and better lipid response to statin therapy did not translate into a risk reduction for myocardial infarction.93 Because statins exert their pleiotropic effects independent of 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibition and lipid lowering, an increased hepatic exposure in these subjects would not necessarily result in a decreased risk for myocardial infarction.93 Nevertheless, knowledge still is limited regarding the importance of these genetic variants in the ABCB1 gene to the cardiovascular outcome in patients receiving statin therapy. Genetic Susceptibility to Statin-Induced Side Effects Statin use has been associated with statin-induced myopathy and rhabdomyolysis. The incidence of statin-induced myopathy varies between 0.01% and 0.4% depending on the dose and type of statin used.94 In contrast, statin-induced rhabdomyolysis is a rare but potentially fatal complication that occurs in fewer than 1 in 10,000 patients.95 Several perioperative factors may be associated with statininduced myopathy. These include length of surgery, concomitant medical therapy, liver dysfunction, renal insufficiency, and hypothyroidism.96 The mechanism of statin-induced myopathy is poorly understood, and procedures for identifying patients at risk have not been developed. However, a variation in a gene on chromosomes 12p12 and 2 that encodes for the organic anion-transporting polypeptide OATP1B1 (SLCO1B1) (Fig 3) may be associated with simvastatin-induced myopathy.97 This polypeptide is responsible for the transport of statins into liver cells, particularly simvastatin and atorvastatin.11 Consequently, a genetic polymorphism of this transporter polypeptide may significantly influence the pharmacokinetics of statins and their lipid-lowering effect and toxicity.82 The SNP of the gene encoding this polypeptide results from the substitution of alanine for valine (Val174Ala) at amino acid position 174. The minor allele frequency of the Ala174 variant is ⬍1% in Yoruba Nigerians and ⬎20% in whites.97 The reported functional consequence of the Ala174 variant is a reduced statin uptake by hepatocytes.98 In clinical studies, patients heterozygous or homozygous for the Ala174 variant had higher plasma concentrations of simvastatin, atorvastatin, and rosuvastatin compared with patients who were homozygous for the low-risk variant (Val174).99,100 As a result, this variant was associated more frequently with severe statininduced myopathy, particularly in patients who received simvastatin.97 However, in regard to perioperative cardiovascular complications, the predictive value and clinical usefulness of these polymorphisms have not been defined. PHARMACODYNAMICS OF STATINS: GENETIC POLYMORPHISMS ASSOCIATED WITH THE PLEIOTROPIC EFFECTS

Genetic Modifiers of Statin Pharmacodynamics: Kinesin-like Protein 6 An SNP of the kinesin-like 6 (KIF6) gene is associated with an altered response to statin therapy and results from an arginine substitution for tryptophan at amino acid position 719 (Trp719Arg).101 The KIF6 gene is located on chromosomes 6p21 and 2, and the polymorphism of this gene is common,

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occurring in 34% to 58% of whites.101,102 Kinesin-like protein 6 belongs to a class of motor proteins involved in the intracellular transport of membrane organelles, protein complexes, and messenger RNA along microtubules.103 The Trp719Arg polymorphism affects the transport of these intracellular elements, likely by changing the binding properties of the kinesin-like protein 6.104 Studies have found that carriers of the Trp719Arg polymorphism were at an increased risk for CAD,101 but when these subjects were treated with statins (pravastatin or atorvastatin), the incidence of cardiovascular complications was reduced significantly.101,102 The observed beneficial effect of statin use was not associated with lowering serum lipid or C-reactive protein levels and was significant as early as 30 days after statin therapy was initiated.102 Contrary to these observations, recent large-scale clinical trials105,106 have found that carriers of the Arg719 variant were not at an increased risk for cardiovascular complications. Furthermore, no consistent benefit was derived from low- or high-dose statin use (ie, rosuvastatin, atorvastatin, and simvastatin) for reducing cardiovascular morbidity and mortality in carriers or noncarriers.106 Finally, carriers of the Arg719 variant did not have a significantly higher incidence of cardiovascular events or significant differences in serum lipid and C-reactive protein levels compared with noncarriers.106 In conclusion, given the inconsistency in findings related to the association of the Trp719Arg polymorphism and statin efficacy for preventing cardiovascular complications, additional studies should be considered to explore the mechanism by which the genetic polymorphism of the KIF6 gene affects the function of kinesin-like protein 6 in the development and prevention of cardiovascular disease.102 Genetic Modifiers of Statin Pharmacodynamics: Toll-like Receptor 4 The toll-like receptor 4 (TLR4) is expressed on cardiomyocytes, macrophages, and endothelial and smooth muscle cells. The gene encoding for TLR4 is located on chromosomes 9q33 and 1. Toll-like receptors have an essential role in recognizing microbial components, but their receptors also may recognize cytokines released during the inflammatory response. The activated toll-like receptors then initiate innate and adaptive immunity.107 Two SNPs of the TLR4 gene have been described that result in amino acid substations in the extracellular domain of the receptor, which is associated with a blunted immunologic108 and proinflammatory response.109 One of these 2 polymorphisms results from a glycine substitution for aspartic acid at amino acid position 299 (Asp299Gly), and the other polymorphism is the result of threonine-to-isoleucine substitution at amino acid position 399 (Thr399Ile). The frequency of a combination of the Gly299 variant and the Ile399 variant is 11%, and the frequency of the Gly299 variant without the Ile399 variant is 1.4% in whites.110 A study by Kiechl et al109 found that subjects with the Gly299 variant with or without the Ile399 variant had less inflammation and lower levels of acute-phase reactants, soluble adhesion molecules, and other mediators of inflammation. Most importantly, patients with the Gly299 polymorphism were at a

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reduced risk of atherosclerosis.109 Recently, the role of the TLR4 polymorphisms in the progression of coronary atherosclerosis and the response to statin therapy was investigated. Boekholdt et al110 found that carriers of the Gly299 variant did not have a significantly lower risk for cardiovascular events compared with noncarriers (29.6% v 18.1%, p ⫽ 0.10). However, when treated with pravastatin, the Gly299 carriers had a lower risk for cardiovascular events than noncarriers (2.0% v 11.5%, p ⫽ 0.045). There was also a greater reduction in risk for cardiovascular events in Gly299 carriers treated with pravastatin (29.6% in the placebo group and 2.0% in the pravastatin group, p ⫽ 0.0002) compared with noncarriers (18.1% in the placebo group and 11.5% in the pravastatin group, p ⫽ 0.03).110 The mechanism by which statins interact with the TLR4 polymorphism and exert their beneficial effect is not fully understood. However, it has been known that statins are associated with reductions in low-density lipoprotein cholesterol and oxidized low-density lipoprotein cholesterol; the latter is a potent upregulator of TLR4. The presence of the Gly299 variant, which attenuates receptor signaling and diminishes the inflammatory response, combined with statin use, results in an inefficient initiation of vascular inflammation, which may lead to reduced coronary artery plaque inflammation and stabilization.110 Genetic Modifiers of Statin Pharmacodynamics: G Protein ␤3-Subunit Key components of the intracellular signaling pathway are G proteins that are composed of ␣-, ␤-, and ␥-subunits. G proteins are activated mainly by G protein– coupled receptors. The gene that encodes for the G protein ␤3-subunit is located on chromosomes 12p13 and 31, and the most significant polymorphism of the gene results from a single base change from cytosine to thymine (C825T).111 The minor allele frequency of the C825T polymorphism varies significantly from 38% in whites to 53% in Chinese and 91% in South Africans.112 The C825T polymorphism is associated with enhanced G protein activity. A study by von Beckerath et al113 found that this polymorphism also is involved with the development of CAD and with the pathophysiologic process involved in atherosclerosis. Furthermore, the functional consequences of the C825T polymorphism are associated with enhanced vasoconstriction and activation of neutrophils and platelets, which could increase the risk for coronary plaque disruption and subsequent myocardial infarction.114 The G protein pathway also plays a role in lipid metabolism, particularly in regulating low-density lipoprotein receptor gene expression in vascular smooth muscle cells.115 Nevertheless, clinical studies on the effect of the C825T polymorphism risk for myocardial infarction have yielded controversial results. Earlier studies reported that the C825T polymorphism is associated with an increased risk for myocardial infarction,114,116 but subsequent studies could not confirm these findings.117,118 However, a more recent case-control study with a large sample size found that homozygous and heterozygous carriers of the C825T polymorphism were hospitalized less frequently for myocardial infarction.119 Furthermore, the greatest reduction in myocardial infarction rate was observed in

KERTAI, FONTES, AND PODGOREANU

patients who used statins and were homozygous or heterozygous carriers of the C825T variant. The mechanism of interaction between the C825T polymorphism and statins is not fully understood. Future studies that combine pharmacogenetics research with characterizing the functional consequences of the C825T polymorphism in the presence of statin therapy may provide a better understanding of the underlying mechanism of this interaction.119 CLINICAL IMPLICATIONS AND CONCLUSIONS

In this review, the authors have summarized the potential implications of pharmacogenetics to individualized perioperative ␤-ADRB and statin pharmacotherapy by highlighting genetic polymorphisms that affect response, safety, and toxicity to these 2 classes of drugs commonly used in the perioperative period. Evidence suggests that genetic polymorphisms in the adrenergic signaling pathway are important risk factors or modifiers of cardiovascular disease. Furthermore, these genetic polymorphisms may influence significantly the pharmacodynamics of ␤-ADRBs in the prevention of cardiovascular complications.12 Overall, ␤1-adrenergic-receptor polymorphisms appear to have the most robust functional consequences affecting the risk for cardiovascular complications during ␤-ADRB therapy. However, important studies relating any of these variants with either efficacy of perioperative ␤-blockade (incidence of perioperative myocardial infarction) or its safety (incidence of perioperative bradycardia/hypotension and stroke) are lacking. Studies that explored genetic factors involved in the biotransformation of ␤-ADRBs revealed that variants in the CYP2D6 isoenzyme are associated with significantly altered ␤-ADRB metabolism and adverse cardiovascular effects.21 Moreover, a significant relationship between ␤1-adrenergic receptor and CYP2D6 polymorphisms that can correlate with dose responses to ␤-ADRBs has been reported.120 These observations suggest that future clinical trials on perioperative ␤-ADRB therapy should include genotype analysis of study subjects to identify any ␤1-adrenergic-receptor polymorphisms and/or CYP2D6 variants. If prospectively proven to be predictive of risk for cardiovascular complications and altered ␤-ADRB metabolism, these biomarkers would provide the basis for informing the currently controversial optimal perioperative ␤-ADRB therapy with increased efficacy and safety.10 Although statin pharmacogenetics has been investigated extensively, clinical studies have found little evidence to support clinical applications. Nevertheless, the identification of genes and their polymorphisms that influence statin responsiveness may help explore the molecular components of lipid metabolism and inflammatory pathways that mediate statin effect.83 There is some evidence that a set of genetic polymorphisms influences the effectiveness of statins for reducing cardiovascular complications101,102,106,110 and also reduces the risk for serious adverse effects, such as statin-induced myopathy (Table 2).11 However, given that multiple genes influence statin efficacy, future clinical studies will require a more comprehensive design combining pharmacogenetic and functional studies to help unravel the mechanisms underlying genetic variation in statin responsiveness as well as identifying clinically useful biomarkers to guide therapy.

PHARMACOGENOMICS OF ␤-BLOCKERS AND STATINS

1111

Table 2. Genetic Polymorphisms Involved in Variable Pleiotropic Responses to Statins Gene Name (Gene Symbol)

Polymorphism

Effect of Polymorphism

Cytochrome P450 2C9 (CYP2C9) and P450 3A4 (CYP3A4) P-glycoprotein transporter (ABCB1)

CYP2C9*2/3 and CYP3A4*4

Decreased activity, higher statin concentration, but effect is not known83 Better lipid response but no effect on cardiovascular risk84,91-93 Higher plasma statin concentration, increased risk of myopathy97,99,100 Probably greater risk reduction for coronary artery disease with statin use101,102 Diminished inflammatory response combined with statin use109,110 Greater risk reduction for myocardial infarction rate with statin use114,116,119

Organic anion-transporting polypeptide (OATP1B1) Kinesin-like 6 (KIF6)

Gly412Gly, Ser89Ala/Tyr, Ile1145Ile Val174Ala Trp719Arg

TLR4

Asp299Gly

G protein ␤3 subunit

C825T

The findings of studies on pharmacogenomics of ␤-ADRBs and statins represent a step forward in the development of a clinical strategy of personalizing prevention and treatment options for perioperative cardiac complications based on genotypes. However, the current approaches to pharmacogenomic research have not provided the level of scientific evidence to meet the expectations of individualized medicine. Many pharmacogenomic studies to date have been underpowered, and the replication of positive results is lacking. Furthermore, most studies only have examined associations of individual genetic polymorphisms with treatment response to drugs. However, differences in drug response are unlikely to be because of any single gene but rather results from complex interactions among multiple

genetic variants and biologic pathways. Therefore, future studies should focus on studying a set of candidate genes and/or apply a genome-wide methodology to study pharmacogenomic associations. Consequently, new approaches are needed to address these challenges, such as a sufficient sample size to analyze a combination of multiple SNPs that influence drug efficacy and the development of novel statistical methods to handle the issue of multiple testing of many SNPs or to study the effects of gene-gene interactions (epistasis). Future pharmacogenomic studies also will require the incorporation of transcriptomics and proteomics to explore the mechanisms underlying pharmacogenomic associations for the prevention and treatment of perioperative cardiac complications.82

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