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the Departments of Anesthesiology (H.P.G., W.D.W., R.W.M., M.P., J.P.M., D.A.S., M.F.N.) and Pharmacology/Cancer Biology (D.A.S.), Duke University Medical Center, Durham, NC
the Department of Statistics (D.M.N.), North Carolina State University, Raleigh.
Abstract
Background and Purpose— Stroke represents a significant cause of morbidity and mortality after cardiac surgery. Although the risk of stroke varies according to both patient and procedural factors, the impact of genetic variants on stroke risk is not well understood. Therefore, we tested the hypothesis that specific genetic polymorphisms are associated with an increased risk of stroke after cardiac surgery.
Methods— Patients undergoing cardiac surgery utilizing cardiopulmonary bypass surgery were studied. DNA was isolated from preoperative blood and analyzed for 26 different single-nucleotide polymorphisms. Multivariable logistic regression modeling was used to determine the association of clinical and genetic characteristics with stroke. Permutation analysis was used to adjust for multiple comparisons inherent in genetic association studies.
Results— A total of 1635 patients experiencing 28 strokes (1.7%) were included in the final genetic model. The combination of the 2 minor alleles of C-reactive protein (CRP; 3'UTR 1846C/T) and interleukin-6 (IL-6; –174G/C) polymorphisms, occurring in 583 (35.7%) patients, was significantly associated with stroke (odds ratio, 3.3; 95% CI, 1.4 to 8.1; P=0.0023). In a multivariable logistic model adjusting for age, the CRP and IL-6 single-nucleotide polymorphism combination remained significantly associated with stroke (P=0.0020).
Conclusions— We demonstrate that common genetic variants of CRP (3'UTR 1846C/T) and IL-6 (–174G/C) are significantly associated with the risk of stroke after cardiac surgery, suggesting a pivotal role of inflammation in post–cardiac surgery stroke.
Key Words: cardiac surgical procedures C-reactive protein interleukin-6 polymorphism stroke
Introduction
Despite considerable advances in cardiac surgery, significant neurological morbidity continues to occur.1–3 Indeed, over the past several decades, many technological advancements in surgery, anesthesia, and the conduct of cardiopulmonary bypass (CPB), coupled with an improved understanding of the pathophysiology of neurological injury, have allowed surgery to be performed on increasingly elderly and high-risk patients.3 Stroke, although less frequent than more subtle types of cerebral injury (such as cognitive dysfunction),2 remains a significant and debilitating complication of cardiac surgery.1 In addition to diminishing quality of life and increasing mortality, stroke following cardiac surgery also increases costs and the use of healthcare resources.1
The variable incidence of post–cardiac surgery stroke is influenced both by patient and procedural risk factors.3 Although many procedural risk factors have been incorporated into stroke risk indices,4 they provide incomplete information regarding the full risks of stroke. These risk indices and associated factors do not include information regarding the genetic makeup of the patient, raising the possibility that heterogeneity seen in the clinical presentation of stroke (both incidence and severity) may partly reflect underlying genotype.
The pathophysiology of stroke in nonsurgical settings involves complex interactions between pathways associated with coagulation, inflammation, lipid metabolism, apoptosis, and direct cellular injury. Within each of these pathways, genetic variants have been identified. Although not exclusively, it appears that proinflammatory and prothrombotic genes may play a significant role in the etiology and outcome after stroke in nonoperative settings.5–11 Consequently, we hypothesized that specific genetic polymorphisms modulate the risk of in-hospital stroke following cardiac surgery.
Materials and Methods
Study Population
The patients enrolled in this study were part of the Perioperative Genetics and Safety Outcomes Study (PEGASUS), an ongoing Institutional Review Board–approved, prospective, longitudinal study at Duke University Medical Center. Patients undergoing coronary artery bypass graft (CABG), valvular, or combined CABG/valve surgery using CPB between March 1996 and May 2002 gave written informed consent to have their clinical and genetic data analyzed in relation to perioperative outcomes.
Clinical Data Collection
Ischemic stroke was the primary outcome variable. All patients with suspected stroke (defined as a new focal abnormality on neurological examination occurring within 1 week of surgery) underwent evaluation by an independent neurologist and confirmatory brain imaging (MRI or CT). Patients with cerebral hemorrhage or generalized encephalopathy were excluded. Individual inclusion criteria were confirmed after independent review of patient hospital records by 2 of the investigators (H.P.G., J.P.M.). Preoperative covariate data including age, sex, race, and preexisting comorbidities considered important to stroke (Table 1) were also recorded.
Candidate Polymorphism Selection
Based on a literature review regarding stroke in experimental, clinical, and cardiac surgical settings, 26 single-nucleotide polymorphisms (SNPs) were identified as primary candidates for analysis. The 26 SNPs represented 13 genes that were classified on the basis of biological function into categories of coagulation, inflammation, and lipid metabolism (Table 2). The coagulation and inflammatory categories were chosen based on extensive literature supporting a role of thrombosis and inflammation in stroke whereas the lipid metabolism category (specifically, apolipoprotein E) was chosen because of its previously demonstrated relationship to other neurological disorders, including post–cardiac surgery cognitive dysfunction.12
Genomic DNA Isolation and Genotype Analysis
Whole blood was collected preoperatively with genomic DNA extraction performed using the Puregene system (Gentra Systems, Minneapolis, Minn). Genotyping assays for SNPs were conducted at Agencourt Bioscience Corporation (Beverly, Mass) by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, using the Sequenom MassARRAY system (Sequenom, San Diego, Calif).13 Primers used and polymorphism details can be foundat http://anesthesia.duhs.duke.edu/pegasus/stroke/1/website_table_1.htm.Using direct sequencing on an ABI3700 capillary sequencer (Applied Biosystems, Foster City, Calif), genotyping reproducibility in this study was validated to be >99% by scoring a panel of 6 polymorphisms in 100 randomly selected patients. Subsequently, genetic results were linked to covariate and phenotypic variables in a relational database.
Statistics
For the association tests, heterozygotes were combined with homozygotes for the minor frequency allele. Consequently, association analyses for each candidate polymorphism were based on 2 genotypic classes, distinguished by the presence of at least 1 minor allele copy.
Fisher exact tests were used to test for independence between stroke and race or gender and between genotype and self-declared race. The presence of cryptic population structure was investigated using 58 unlinked, noncandidate SNPs to fit a hierarchical population structure model.14
For each of the 26 SNPs, 2 tests for independence between stroke and genotype were computed. Tests for genetic effects of SNP pairs were investigated separately within each functional category by distinguishing genotypes bearing at least 1 minor allele at both SNPs. This genotype partitioning yielded a 2x2 table of 2-SNP genotype by stroke, thereby allowing a test for independence with individual SNP pairs. Robustness of the genetic effect was examined using logistic regression modeling adjusting for age, and in 3-factor models including genotype, age and either -blocker therapy,15 race, or gender. For the multivariable model, race was characterized as white/nonwhite.
Because our analysis strategy involved many separate tests of independence, thus accounting for multiple testing, we used random permutation analysis to adjust probability values.16 We generated 5000 copies of our data set, randomly reassigning stroke events to study subjects, thereby disassociating genotype from stroke events. For each permutation, probability values were calculated for a genotype partition of each SNP pairs. For each genotype partition, the smallest probability value was retained to estimate the distribution of 5000 "smallest" probability values under the null hypothesis. An adjusted probability value was computed as the fraction of permutation probability values that were less than the observed probability value. All statistical analysis was performed using SAS and SAS/Genetics version 8.02 (SAS Inc).
Results
Demographics and intraoperative characteristics of the patients are presented in Table 1; with the exception of age and preoperative -blocker therapy, there were no differences between those with or without stroke. Of the 2104 patients enrolled, 38 (1.8%) experienced in-hospital stroke. Because not all SNPs were available in all patients, a subset of patients with complete clinical and genetic data were used for analysis. The final multivariable genetic model included 1635 patients, of whom 28 (1.7%) experienced stroke.
No single SNP alone was significantly associated with stroke. However, a SNP pair representing individuals having minor alleles for C-reactive protein (CRP; 3'UTR 1846C/T) and interleukin (IL)-6 (–174G/C) was significantly associated with stroke. Of the 1635 patients, 583 (36%) had minor alleles for both SNPs. The incidence of stroke in patients with this genotype was 3.09% (18/583), compared with 0.95% (10/1052) in patients without this combination (odds ratio [OR], 3.3; 95% CI, 1.4 to 8.1; exact P=0.0023; Figure). The 2 SNPs involved were among the 9 in the inflammation category. After adjustment for multiple comparisons (involving 36 pair-wise combinations) using permutation testing, the probability value for this SNP combination remained significant (adjusted P=0.023).
Incidence of postoperative stroke in patients possessing the combination of SNPs in both CRP (3'UTR 1846C/T) and IL-6 (–174G/C). This SNP combination significantly increased the risk of stroke (OR, 3.3; 95% CI, 1.4 to 8.1; P=0.0023).
Because of the relatively few stroke events, we were limited in the additional terms that could be tested in a multivariable model. However, as the stroke patients were older, a second model was constructed that included age as a covariable. That model remained significant (P=0.0001) for the SNP combination (OR, 3.2; 95% CI, 1.4 to 7.8; P=0.0020), and age (OR, 1.05 for each additional year; 95% CI, 1.02 to 1.10; P=0.0057). When -blocker therapy was included as a covariable, it became nonsignificant (P=0.5015), whereas the SNP combination remained significant (P=0.0034).
With regard to possible gender or population structure effects, there was no association between stroke and gender (P=0.4933) nor between stroke and self-declared race (P=0.3009; Table 3). Although several genotypes were differentially distributed among races, neither race nor gender was associated with stroke (P=0.2083 and P=0.6437, respectively). Furthermore, the ethnic indicator variables from the population structure analysis were similarly nonsignificant when added to the SNP combination and age in the model (P=0.1910).
Discussion
Stroke after cardiac surgery is a relatively uncommon but potentially devastating event. Previous investigations designed to understand stroke risk factors have typically relied on clinical variables to construct risk indices.4 Age, for example, is a robust risk factor for perioperative stroke and was reconfirmed in this present study.4 We now demonstrate a new significant relationship, independent of age, between stroke and the genetic makeup of an individual. Our findings reveal that the concurrent presence of at least 1 minor allele at each of 2 loci (CRP: 3'UTR 1846C/T; IL-6: –174G/C) is a risk factor for stroke, increasing risk more than 3-fold.
The observation that the interaction of these 2 inflammatory SNPs contributes to perioperative stroke suggests that inflammatory pathways may be important mechanistic factors in either initiating or otherwise modulating stroke after cardiac surgery. This interpretation is consistent with current knowledge regarding CPB initiating and IL-6 mediating a robust inflammatory response.17 This finding is also consistent with the view that inflammation plays an important role in the etiology of stroke in the general population.8,14 Unexpectedly, thrombotic polymorphisms, significantly associated with stroke in non–cardiac surgery settings,10,11 were not shown to be related to stroke in our analysis, suggesting that inflammatory etiologies may supersede thrombotic causes of stroke in cardiac surgery patients.
The 2 inflammatory polymorphisms identified have been previously characterized outside the setting of cardiac surgery. The polymorphism involving the promoter region of the IL-6 gene (–174G/C) has been shown to be an independent risk factor for lacunar stroke.18 In addition to increasing stroke incidence, the same SNP has been shown to increase stroke severity at 3 months. Interestingly, the IL-6 SNP, when combined with a second inflammatory polymorphism (ICAM-1, 469E/K), appears to have an even greater effect on stroke risk. Pola et al demonstrated that both IL-6 and ICAM-1 SNPs were independently associated with stroke risk (OR, 8.6 and 4.0, respectively) but when present together had a synergistic effect (OR, 10.1; 95% CI, 2.1 to 48.5).7 Following cardiac surgery, the IL-6 polymorphism is thought to influence plasma levels and functional activity of the IL-6 protein and has been associated with other perioperative inflammatory complications.19,20 However, association between IL-6 polymorphisms and perioperative neurological outcome has not been previously described.
CRP, an acute-phase reactant implicated in inflammation, has repeatedly been shown to influence the risk of cardiovascular disease and stroke.9 Indeed, Rost et al demonstrated that elevated plasma CRP significantly predicts risk of stroke and transient ischemic attack in the elderly.9 The C/T polymorphism in the 3'UTR of the CRP gene has been associated with increased basal plasma levels of CRP and is part of a haplotype associated with elevated peak CRP levels after CABG.20,21 The mechanism by which this variant might influence CRP expression remains to be established, but it may involve alterations in the stability of the CRP mRNA which depend on the 3'UTR sequences.22 By further increasing CRP levels, this polymorphism may increase the risk of stroke, although the precise mechanism by which CRP may act on stroke in this setting is not well understood.23
Of particular interest is our finding that it is the interaction between IL-6 and CRP SNPs, and not each individual SNP alone, that appears to be an important determinant of stroke risk after cardiac surgery. Epistasis, or the interaction between genes, is a phenomenon of increasing interest in genetic epidemiology.24 A plausible biological model could be postulated for the epistasis event observed in the current study, as induction of the acute phase CRP response in hepatocytes is promoted by the synergistic action of proinflammatory cytokines IL-6 and IL-1.25 Therefore, genetic variants modulating IL-6 levels and the expression of CRP may contribute to the perioperative proinflammatory phenotype seen in cardiac surgical patients.
There are some potential limitations to this study. As with all association studies, one must be cautious to avoid an inference of causation when interpreting the SNP combination–stroke link. In addition, limitations of multiplex genotyping resulted in missing genotypes for some SNPs in some patients. These data are randomly missing with respect to patient characteristics and, therefore, would not be expected to bias tests of association. Population admixture is also a concern in genetic association studies because it can mask, or falsely identify, a phenotype with a genetic trait.26 Importantly, we identified no significant association of race to stroke, thereby minimizing this possible confound. Lastly, although we speculate that this unique gene combination may have mediated its stroke effect via changes in IL-6 and CRP protein levels, we did not measure these in our patients. However, these SNPs, at least individually, have been previously demonstrated in cardiac surgery patients to influence their respective protein levels.20,21
The implications of these findings are several-fold. Better understanding stroke risk in cardiac surgery settings should facilitate informing patients of enhanced individual risk. Because this SNP combination occurs in a large percentage of patients, 36% in this study, such knowledge has widespread relevance. In addition, perioperative screening could facilitate alterations in the usual course of the surgical procedure with implementation of procedures designed to reduce this risk (eg, absence of aortic cross-clamping). In addition, when new neuroprotective strategies and/or related pharmacological agents become available, a more rational allocation of these likely higher-cost therapies to the highest-risk patients might be facilitated. Most importantly, identification of this unique polymorphism combination emphasizes the significant effect that inflammation potentially plays in cardiac surgery–related stroke. Identifying specific mechanisms whereby this SNP combination mediates its effects will be important in understanding targets whereby modulating perioperative inflammation might result in improvement in outcome after cardiac surgery.
Acknowledgments
This study was funded, in part, by National Institutes of Health grant AG17556 (to D.A.S.) and American Heart Association Grants 0120492U (to M.V.P.), 0256342U (to J.P.M.), and 9970128N (to M.F.N.); and the Duke Clinical Research Centers Program (National Institutes of Health grant M01-RR-30). D.A.S. is a senior fellow in the Duke Center for the Study of Aging and Human Development. We thank Dr Larry B. Goldstein for helpful scientific comments on the manuscript and Cheryl Stetson and Zarrin Brooks for help with manuscript preparation and administrative details.
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