A Prospective Evaluation of the CD14 and CD18 Gene Polymorphisms and Risk of Stroke
Background and Purpose— Genetic polymorphisms of the CD14 lipopolysaccharide receptor gene (CD14) and the CD18 leukocyte adhesion molecule gene (CD18) have recently been hypothesized to be risk factors for atherothrombosis. However, no prospective data on subsequent risk of stroke are available. The present investigation was conducted to examine the possible association between the CD14 C(−260)T and CD18 codon 441 gene polymorphisms and the incidence of stroke in a large, prospective, matched case-control sample from the Physicians’ Health Study.
Methods— In the Physicians’ Health Study, 14 916 apparently healthy men were followed over a 12-year period for stroke. Using a nested case-control study design, 338 study participants who developed stroke (cases) and 338 age- and smoking-matched study participants who remained free of reported disease during follow-up (controls) were evaluated. Both polymorphisms were determined by polymerase chain reaction with subsequent and respective restriction fragment length polymorphism gel electrophoresis.
Results— All observed genotype frequencies were in Hardy-Weinberg equilibrium. The allele and genotype distributions of the polymorphisms tested were similar among cases and controls, such that the relative risk of future stroke was 0.87 for CD14 C(−260)T (95% CI=0.69 to 1.11; P=0.27) and 0.99 for CD18 codon 441 (95% CI=0.77 to 1.28; P=0.96) assuming an additive mode of inheritance. No evidence of association was observed assuming dominant or recessive model, and similar null results were observed in subgroup analysis restricted to thromboembolic events
Conclusions— In this large, prospective study, we found little evidence that the two previously described polymorphisms in the CD14 and CD18 genes are associated with risks of future stroke.
Abnormalities in cell-mediated immune/inflammatory response have been observed in the early stages of stroke.1 Among the key mediators of this process are monocyte differentiation antigen CD14 (CD14) and leukocyte adhesion molecule CD18 (CD18). Although an increase in monocyte CD14 and neutrophil CD18 expression has been observed after acute ischemia,2–4⇓⇓ the underlying mechanisms of this effect remain poorly understood.
Recently, the T allele at position −260 of the CD14 monocyte differentiation antigen gene (CD14)5,6⇓ and a codon 441 polymorphism in exon 11 of the CD18 leukocyte adhesion molecule gene (CD18)7 have been hypothesized to be risk factors for the pathogenesis of atherothrombosis. These data, however, were derived from retrospective studies, and thus require direct testing in prospective cohorts.8 We, therefore, investigated the role of CD14 C(−260)T polymorphism as a risk marker for stroke in the Physicians’ Health Study (PHS), a large-scale, prospective cohort of apparently healthy middle-aged men. Based on the potential role of CD18 in atherothrombosis, we simultaneously evaluated a CD18 codon 441 polymorphism in this data set.
Subjects and Methods
We used a nested case-control design within the PHS, a randomized, double-blinded, placebo-controlled trial of aspirin and beta-carotene initiated in 1982 among 22 071 male, predominantly white, US physicians, 40 to 84 years of age at study entry.9 Before randomization, 14 916 participants provided an ethylenediaminetetraacetic acid-anticoagulated blood sample from which the buffy coat was separated and stored for genetic analysis. All participants were free of prior myocardial infarction, stroke, transient ischemic attacks, and cancer at study entry. Yearly follow-up self-report questionnaires provide reliable updated information9 on newly developed diseases and the presence or absence of other cardiovascular risk factors. History of cardiovascular risk factors, such as hypertension, diabetes, or hypercholesterolemia, was defined as subjects who had or were on medication for the condition(s) at entry into the study.
Stroke was defined by the presence of a new focal neurological deficit, with symptoms and signs persisting for >24 hours, and was ascertained from blinded review of medical records and autopsy results. Stroke was classified as thromboembolic, subarachnoid hemorrhage, intraparenchymal hemorrhage, or other, on the basis of clinical reports, CT scan, or MRI scanning. For the present investigation, 338 incident cases of stroke were identified among those study participants who had stored blood samples.
For each case, a control matched by age, smoking history, and length of follow-up was chosen among those subjects who remained free of vascular diseases. The study was approved by the Brigham and Women’s Hospital Institutional Review Board for Human Subjects Research.
Details of genotype determination for the CD14 C(−260)T polymorphism have been described previously.10 Genotypes for codon 441 in exon 11 of CD18 were determined by a technique using polymerase chain reaction with subsequent and respective restriction fragment length polymorphism. Reagent concentrations in the 15-μL polymerase chain reaction were 330 nmol/L each for sense (5′-CGG CCA CAG AGT GCA TCC AGG A-3′) and reverse (5′-GCC GCA CTC CAA GAA GCC CTT G-3′) primers, 166 mmol/L for deoxynucleotide triphosphates, 2.0 mmol/L for MgCl2, and 0.15 U of Taq polymerase. Samples were amplified for 36 cycles consisting of denaturation at 94°C for 30 seconds, annealing at 62°C for 45 seconds, and extension at 72°C for 1 minute, followed by a final extension step at 72°C for 5 minutes. The T and C alleles were identified by subsequent AvaII restriction digest, electrophoretic size fractionation, and ethidium bromide visualization.7 Allele T represents the absence of an AvaII restriction site; allele C, presence of an AvaII site.7 To confirm genotype assignment, the genotyping procedure was performed on all samples on two separate occasions. Disagreements (<1% of all scoring) were resolved by a joint reading, and where necessary, a repeat genotyping reaction. Results were scored blinded as to case-control status.
Allele and genotype frequencies among cases and controls were compared with values predicted by the Hardy-Weinberg equilibrium using the χ2 test. Relative risks of stroke associated with each genotype, with 95% CI, were calculated by means of logistic regression models conditioning on the matching of age and smoking status, and further controlled for randomized treatment assignment and history of hypertension and diabetes. All probability value are 2-tailed.
Baseline characteristics of stroke cases and controls are shown in Table 1. As expected, the case participants had a higher prevalence of hypertension and diabetes at baseline as compared with controls. Age and smoking status were identical in the 2 groups as a result of matching.
Table 2 shows the allele and genotype frequencies for the study population, which are consistent with that predicted by the Hardy-Weinberg equilibrium. As also shown in Table 2, the allele and genotype frequencies were similar among cases and controls such that no overall difference in genotype distribution was observed (χ22df=2.54, P=0.28 for CD14 C(−260)T; χ22df=0.45, P=0.80 for CD18 codon 441). Thus, the relative risk of future stroke was 0.87 (95% CI=0.69 to 1.11; P=0.27) for CD14 C(−260)T and 0.99 (95% CI=0.77 to 1.28; P=0.96) for CD18 codon 441 (Table 3), assuming an additive mode of inheritance. Similar null results were observed assuming a recessive or a dominant model. These effects were virtually identical in a subgroup analysis limited to thromboembolic stroke (0.91, 95% CI=0.69 to 1.19, P=0.49 for CD14 C(−260)T; 0.94, 95% CI=0.70 to 1.26, P=0.68 for CD18 codon 441; assuming an additive model). In further stratification, similar null effects were observed for those events classified by the end points committee solely as embolic (CD14 C(−260)T: 1.13, 95% CI=0.64 to 1.99, P=0.68; CD18 codon 441: 0.66, 95% CI=0.33 to 1.31, P=0.23), as well as those classified solely as thrombotic events (CD14 C(−260)T: 0.93, 95% CI=0.57 to 1.52, P=0.77; CD18 codon 441: 0.92, 95% CI=0.53 to 1.61, P=0.78), assuming an additive model.
In this large, prospective investigation, we found no significant evidence of association between the CD14 C(−260)T or CD18 codon 441 polymorphism and risk of stroke, either in the total cohort or in subgroup analysis limited to thromboembolic events.
The current null data conflict with two prior retrospective studies,5,6⇓ which suggested the CD14 C(−260)T polymorphism as a risk factor for atherothrombotic disease. However, on close examination of one of the prior studies, it is apparent that the association observed5 may have been a result of an overestimation of allele frequency based on an inadequate number of control subjects, an issue that can lead to erroneous estimates of background mutation rates.8 In the other prior positive study, the observed association was found only in a subgroup analysis of 76 subjects with no history of hypertension, who were nonsmokers and aged >62 years.6 Because no significant association was found in the overall cohort of 2228 men,6 it is possible that overdependence on post hoc subgroup analysis may have led to the apparent false-positive finding.8 By contrast, our null data for stroke are consistent with a recent study examining the same CD14 polymorphism in a Japanese population,11 as well as in our own study of myocardial infarction.10
Potential limitations of the current data should be considered. The participants of our study were almost exclusively Caucasian men, and thus these data should not be construed to rule out a possible association in other ethnic groups. However, unlike prior studies, our cases and controls derive from a homogeneous, prospective population, and thus our study design greatly reduces the potential for selection bias and confounding by ethnicity. In addition, because the overall rate of stroke in the PHS cohort is low, the ability to detect risk in this population may be limited. However, prior studies have documented the ability of the present study design to detect increased genetic risk in these individuals.12–14⇓⇓ Further, based on the size of our study, we had the ability to detect, with 80% probability, at an alpha of 0.05, a risk ratio of >1.20 for an association of the T allelic variant of CD14 with stroke and a risk ratio of >1.25 for the T allele of CD18, assuming an additive model. Nonetheless, we recognize that the number of hemorrhagic events in our study is small and inadequate to address a potential true effect for this end point.
It is important to recognize that association studies can only examine the possible association between phenotype and the tested polymorphism. Our study therefore cannot exclude the possibility that examination of a different polymorphism (which would by definition have to be in linkage disequilibrium with the one tested) might obtain different results. However, in this large, prospective, nested case-control study among middle-aged US men, we found little evidence of association between the CD14 C(−260)T or CD18 codon 441 polymorphism and risk of thromboembolic stroke.
This study was supported by the National Heart, Lung, and Blood Institute (HL58755, HL63293), the American Heart Association, and the Doris Duke Charitable Foundation (P.M.R.).
- Received October 1, 2001.
- Revision received January 18, 2001.
- Accepted January 21, 2001.
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- ↵Hubacek JA, Pit′ha J, Skodova Z, Stanek V, Poledne R. C(−260)→T polymorphism in the promoter of the CD14 monocyte receptor gene as a risk factor for myocardial infarction. Circulation. 1999; 99: 3218–3220.
- ↵Unkelbach K, Gardemann A, Kostrzewa M, Philipp M, Tillmanns H, Haberbosch W. A new promoter polymorphism in the gene of lipopolysaccharide receptor CD14 is associated with expired myocardial infarction in patients with low atherosclerotic risk profile. Arterioscler Thromb Vasc Biol. 1999; 19: 932–938.
- ↵Steering committee of the physicians’ health study research group. Final report of the aspirin component of the ongoing physicians’ health study. N Engl J Med. 1989; 312: 129–135.
- ↵Ito D, Murata M, Tanahashi N, Sato H, Sonoda A, Saito I, Watanabe K, Fukuuchi Y. Polymorphism in the promoter of lipopolysaccharide receptor CD14 and ischemic cerebrovascular disease. Stroke. 2000; 31: 2661–2664.
- ↵Ridker PM, Hennekens CH, Miletich JP. G20210A mutation in prothrombin gene and risk of myocardial infarction, stroke, and venous thrombosis in a large cohort of US men. Circulation. 1999; 99: 999–1004.
- ↵Rubattu S, Ridker P, Stampfer MJ, Volpe M, Hennekens CH, Lindpaintner K. The gene encoding atrial natriuretic peptide and the risk of human stroke. Circulation. 1999; 100: 1722–1726.