Abstract
Variations in candidate genes participating in oxidative stress, inflammation and their interactions are potentially associated with diseases of atherosclerotic origin. We investigated independent and joint associations of variations in cholesterol ester transfer protein (CETP), interleukin 8 (IL8), peroxisome proliferator activator receptor alpha (PPARA) and toll-like receptor 4 (TLR4) genes with incident non-fatal myocardial infarction (MI) or ischemic stroke. In a population-based case-control study, cases (848 MI and 368 ischemic stroke) and controls (2682) were recruited from postmenopausal women and hypertensive men/women who were members of Group Health in Western Washington State. Common tag single nucleotide polymorphisms (n=34) representing gene-wide variations were selected from gene sequencing data using pairwise linkage disequilibrium. Haplotypes were inferred using a modified expectation maximization algorithm. Multivariate logistic regression evaluated individual haplotype and SNP-disease associations in log-additive models. Global haplotype tests assessed overall gene-disease associations. Logic regression was used to evaluate gene-gene interactions. False discovery rates and permutation tests were used for multiple testing adjustment in evaluating independent associations and interactions respectively. Overall, gene-wide variations in PPARA and TLR4 genes were associated with MI. The minor allele of the PPARA SNP, rs4253623, was associated with a higher risk of MI (odds ratio: 1.25, 95%CI: 1.08–1.46) while the minor allele of the TLR4 SNP, rs1927911, was associated with a lower risk of MI (odds ratio: 0.88, 95%CI: 0.77–0.99). No within gene or gene-gene interaction was associated with MI or ischemic stroke risk. Potential SNP-disease associations identified in the current study are novel.
Keywords: genetic risk factors, myocardial infarction, ischemic stroke
Introduction
Oxidative stress, inflammation and their interactions are potentially associated with diseases of atherosclerotic origin.1 Cholesterol ester transfer protein (CETP), interleukin 8 (IL8), peroxisome proliferator activator receptor alpha (PPARA) and toll like receptor 4 (TLR4) have been shown to play roles in oxidative stress, inflammation and cardiovascular diseases (CVD).2–6 Supporting evidence for these functions has led investigators to hypothesize the role of their genetic counterparts in diseases such as myocardial infarction (MI) and stroke. Previous studies evaluating associations between genetic variations in CETP, IL8, PPARA and TLR4 with MI and/or stroke reported inconsistent findings warranting further comprehensive replication studies.5, 7–10 Most studies did not evaluate whole gene variations and were typically conducted among patients with demonstrated atherosclerotic lesions to assess disease progression and outcome. Further, no population-based study to our knowledge investigated global interactions between these four genes in relation to MI and/or stroke. We conducted a large population-based case-control study to evaluate independent and joint associations of gene-wide variation in CETP, IL8, PPARA and TLR4 with non-fatal incident MI or ischemic stroke.
Methods
The study was part of a group of case-control studies that investigate risk factors associated with cardiovascular diseases. Study participants (Table 1) were selected from members of Group Health, a large integrated health care organization in Washington State. Cases were nonhypertensive postmenopausal women and hypertensive (pharmacologically treated) men and women, 30–79 years of age who suffered incident non-fatal MI or ischemic stroke during the study period (January 1, 1995 through December 31, 2002). Cases were identified using computerized discharge diagnoses from Group Health facilities and from claims databases. The index date for cases was the date of admission for the first MI or ischemic stroke. Incident events of MI were verified based on symptoms, electrocardiogram findings, enzyme levels, and physician diagnosis and treatment. Ischemic stroke diagnosis was verified using history of rapid onset of a neurologic deficit that persisted at least 24 hours or evidence of infarction on brain imaging studies or at surgery. Cases whose index event was a complication of a procedure were not eligible for the study. Controls were a stratified random sample of Group Health members selected from enrollment files based on person-time and were frequency matched to MI cases by age (within decade), sex, treated hypertension status and calendar year of index date. The index date for controls was a random computer generated date within the matching calendar year. Both cases and controls were alive at time of recruitment and had at least 4 visits to a Group Health provider prior to the index date. After exclusion of both cases and controls with previous history of either MI or stroke, a total of 1216 cases (848 MI and 368 IS) and 2682 controls were available for the study. Study eligibility, participant characteristics and risk factor information was collected by medical record review and telephone interview. Information was collected only for the period before the index date. A venous blood sample was collected from subjects and DNA was extracted from white blood cells using standard phenol extraction methods. Group Health Human Subjects Review Committee approved the study and all participants provided written informed consent.
Table 1.
Study population characteristics
| Cases | |||
|---|---|---|---|
| Characteristics | Controls (n=2682) | Myocardial infarction (n=848) | Ischemic stroke (n=368) |
| Men | 41.6 % | 42.8 % | 31.4 % |
| Mean Age (years) | 65.2 | 65.8 | 68.5 |
| Whites | 91.1 % | 91.1 % | 90.8 % |
| Blacks | 3.8 % | 3.7 % | 6.0 % |
| Other | 5.1 % | 5.1 % | 3.2 % |
| Smoker | |||
| Never/Non smoker | 46.2 % | 37.1 % | 50.8 % |
| Former | 43.8 % | 44.6 % | 36.5 % |
| Current | 10.0 % | 18.3 % | 12.7 % |
| Treated Hypertension | 73.1 % | 71.6 % | 71.4 % |
| Treated Diabetes Mellitus | 11.4 % | 24.1 % | 24.3 % |
| Mean Body Mass Index (kg/m2) | 29.5 | 30.1 | 30.0 |
Single nucleotide polymorphisms (SNPs) were identified by genomic resequencing by Seattle Program for Genomic Applications (for CETP, IL8 and PPARA) or Innate Immunity (for TLR4). Subsets of gene-specific tag-SNPs that describe common (minor allele frequencies ≥ 5%) gene-wide variation in European Americans and African Americans were selected using LDselect software that is based on linkage disequilibrium (threshold of correlation r2 = 0.64).11 A total of 50 tag-SNPs (16 CETP, 5 IL8, 9 PPARA and 20 TLR4) representing gene-wide variations were selected for genotyping. Genotyping for 44 of the 50 tag-SNPs (13 CETP, 5 IL8, 7 PPARA and 19 TLR4) was successfully performed by Illumina® with a GoldenGate custom panel using BeadArray® technology. Among these tag-SNPs, 99.9 % of nucleotide pairs were called. After dropping 10 tag-SNPs that were rare (<2.5% minor allele frequency) in the study population, a total of 34 SNPs (13 CETP, 3 IL8, 6 PPARA and 12 TLR4) remained for investigation (Table 2). We used Phase v2.0 to infer haplotypes.12 Haplotype uncertainty was estimated using the diplotype probability. Within a gene, haplotypes with frequencies less than 2.5% among controls were grouped together in a gene-specific haplotype bin assigned “Others”. A total of 23 (6 CETP, 4 IL8, 6 PPARA and 7 TLR4) common haplotypes were investigated.
Table 2.
Single nucleotide polymorphisms (SNPs) and haplotypes
| SNPs | Haplotypes | |||||
|---|---|---|---|---|---|---|
| Gene | Reference number | Position* | MAF** | Haplotype ID | Structure*** | Freq** |
| CETP | rs17231506 | 5’F | 30 % | CETP-A | CGCACGGTCACCT | 20 % |
| rs4783961 | 5’F | 49 % | CETP-B | TACCTGGTCGTCT | 14 % | |
| rs17245715 | 5’F | 10 % | CETP-C | CACATATTCGTCT | 6 % | |
| rs1800775 | 5’F | 49 % | CETP-D | CGTCTGGTCGCCT | 7 % | |
| rs708273 | In2 | 30 % | CETP-E | TACCTGGTCGCGT | 6 % | |
| rs12720922 | In2 | 18 % | CETP-Others§ | --------------------------- | 47 % | |
| rs9939224 | In2 | 21 % | ||||
| rs11076174 | In2 | 8 % | ||||
| rs5883 | Ex9S | 5 % | ||||
| rs4784744 | In10 | 35 % | ||||
| rs1800774 | In12 | 35 % | ||||
| rs289742 | 3’F | 13 % | ||||
| rs12720917 | 3’F | 15 % | ||||
| IL8 | rs4073 | 5’F | 48 % | IL8-A | ATC | 51 % |
| rs2227307 | In1 | 47 % | IL8-B | TGT | 42 % | |
| rs2227543 | In3 | 42 % | IL8-C | TGC | 3 % | |
| IL8-Others§ | ----- | 3 % | ||||
| PPARA | rs4253623 | In2 | 13 % | PPARA-A | GGGCAT | 29 % |
| rs135543 | In2 | 29 % | PPARA-B | AAGTGT | 22 % | |
| rs4253655 | In2 | 16 % | PPARA-C | GAGCGC | 11 % | |
| rs4253699 | In2 | 20 % | PPARA-D | GGATGT | 9 % | |
| rs4253701 | In2 | 10 % | PPARA-E | AAGTGT | 6 % | |
| rs4253765 | In6 | 19 % | PPARA-Others§ | ------------ | 23 % | |
| TLR4 | rs2770150 | 5’F | 27 % | TLR4-A | GCTGCACCGAGT | 27 % |
| rs1927914 | 5’F | 36 % | TLR4-B | ACTGCACCGAGT | 22 % | |
| rs7864330 | In1 | 6 % | TLR4-C | ACTGCACCCAGT | 14 % | |
| rs1927911 | In1 | 28 % | TLR4-D | ATTACACCGAGT | 7 % | |
| rs11536878 | In3 | 12 % | TLR4-E | ATTAAACCGGGT | 6 % | |
| rs4986790 | Ex4N | 6 % | TLR4-F | ATGGCGTCGAGC | 5 % | |
| rs4986791 | Ex4N | 5 % | TLR4-Others§ | --------------------------- | 18 % | |
| rs7869402 | Ex4U | 4 % | ||||
| rs11536889 | 3’F | 15 % | ||||
| TLR4-17050 | 3’F | 16 % | ||||
| TLR4-17723 | 3’F | 5 % | ||||
| rs1927906 | 3’F | 10 % | ||||
SNP position descriptions: F=flanking, Ex=exon, In=intron, N=non-synonymous, U= untranslated.
SNP minor allele frequencies (MAF) and probability-weighed haplotype frequencies among controls.
Haplotype structures in order of SNP list.
Others, a haplotype group that includes all other haplotypes with frequencies less than 5%.
Specific global gene-disease associations were evaluated separately for MI and ischemic stroke using a logistic regression model that included all common haplotypes of the gene and the most common haplotype as a referent. The null hypothesis that no single haplotype odds ratio is different from 1 was used as the global test of association. This global Wald test is not affected by the choice of the referent group. Odds ratios represent the odds of an event associated with each additional copy of the haplotype relative to an additional copy of the referent haplotype. Diplotype probabilities were used as a weight in analyses clustered on participant identifier. The false discovery rate (FDR) correction, q-value, was used for multiple testing (8 global gene tests) correction.13–14 Since genes evaluated were candidate genes previously described, a q-value below 0.20, corresponding to a 20% false discovery rate (FDR), was selected as a threshold for acceptable level of significance.13 In a secondary analysis, separate SNP-specific logistic models were fit for each outcome separately. These models included a single variable for each SNP (additive effect of SNPs coded 0, 1 and 2 for minor allele copies). We did not correct for multiple testing in these analyses.
Logic regression, a method of identifying significant combinations among all 34 SNPs associated with higher or lower risk of MI or ischemic stroke, was used to evaluate both within gene and gene-gene interactions.15 Logic regression identifies Boolean combinations of binary predictors using an adaptive algorithm that selects a combination that minimizes the residual sum of squares or the deviance. Model selection was optimized using cross-validation while multiple testing adjusted significance was evaluated using permutations in the logic regression algorithm. We conducted sensitivity analyses by restricting participants to only whites (to account for population stratification) and also by combining MI and ischemic stroke cases into a larger case group (to increase power). Further, we conducted a sensitivity analysis to assess potential heterogeneity of SNP-disease associations by gender, diabetes status, smoking, obesity and alcohol intake. All confidence intervals were calculated at the 1-alpha = 95% level. All models were adjusted for age, sex, race, index year and treated hypertension status.
Results
Among 3898 study participants included in this study, 41% were male and 91% were white (Table 1). Global gene variation of PPARA was associated with risk of MI (global Wald test p-value=0.033; false discovery rate=13%) (Table 3). Each additional copy of the PPARA-C haplotype was associated with an 82% higher MI risk compared with an additional copy of the referent haplotype. Each additional copy of rs4253623 (part of the PPARA-C haplotype) minor allele was associated with a 25% higher MI risk (Adj. OR: 1.25, 95%CI: 1.08–1.46, p-value: 0.004). Similarly, global gene variation of TLR4 was associated with MI risk (global Wald test p-value=0.008; false discovery rate=6%). Each additional copy of the TLR4-D haplotype was associated with a 40% lower risk of MI compared with an additional copy of the referent haplotype. Minor allele of rs1927911, part of the TLR4-D haplotype, was associated with a 12% lower MI risk (Adjusted OR: 0.88, 95%CI: 0.77–0.99, p-value=0.043). The global CETP gene and MI risk association test was marginal (global Wald test p-value=0.083). However, no common haplotype or SNP of CETP was associated with MI risk. IL8 gene-wide variation was not associated with risk of MI. Global variations in CETP, IL8, PPARA or TLR4 were not associated with ischemic stroke. However, in secondary analysis, minor alleles of two CETP SNPs, rs12720922 and rs9939224, were associated with a similar 24% (Adjusted OR: 1.24, 95%CI: 1.03–1.51, p-value=0.026) and 26% (Adjusted OR: 1.26, 95%CI: 1.05–1.51, p-value= 0.025) higher ischemic stroke risk respectively.
Table 3.
Global gene and haplotype associations with MI and ischemic stroke
| Genes (haplotypes) | Freq (%)** Control/MI/IS | Myocardial infarction (MI) OR** (95%CI) | Ischemic stroke OR** (95%CI) |
|---|---|---|---|
| CETP | |||
| CETP-A | 20/18/19 | Referent | Referent |
| CETP-B | 14/16/13 | 1.19 (0.97–1.45) | 0.84 (0.64–1.10) |
| CETP-C | 6/5/7 | 0.92 (0.69–1.22) | 0.94 (0.65–1.35) |
| CETP-D | 7/6/5 | 0.95 (0.70–1.27) | 0.77 (0.52–1.14) |
| CETP-E | 6/7/6 | 1.29 (0.99–1.68) | 1.02 (0.68–1.49) |
| CETP-Others | 47/48/50 | 1.21 (0.99–1.46) | 0.87 (0.68–1.15) |
| Global Wald test (p-value)§ | 0.083 | 0.621 | |
| False discovery rate (q-value) | 22% | >50% | |
| IL8 | |||
| IL8-A | 51/53/52 | Referent | Referent |
| IL8-B | 42/41/42 | 0.92 (0.78–1.07) | 0.98 (0.78–1.22) |
| IL8-C | 3/3/3 | 0.99 (0.64–1.54) | 0.79 (0.44–1.40) |
| IL8-Others | 3/2/4 | 1.08 (0.51–2.30) | 1.18 (0.54–2.61) |
| Global Wald test (p-value)§ | 0.734 | 0.812 | |
| False discovery rate (q-value) | >50% | >50% | |
| PPARA | |||
| PPARA-A | 29/28/27 | Referent | Referent |
| PPARA-B | 22/20/21 | 1.00 (0.80–1.25) | 1.03 (0.84–1.27) |
| PPARA-C | 11/19/13 | 1.82 (1.20–2.77) | 1.20 (0.92–1.57) |
| PPARA-D | 9/9/7 | 1.08 (0.88–1.33) | 0.83 (0.61–1.12) |
| PPARA-E | 6/5/7 | 0.99 (0.75–1.32) | 1.25 (0.83–1.89) |
| PPARA-Others | 23/19/25 | 1.15 (0.79–1.68) | 1.08 (0.63–1.87) |
| Global Wald test (p-value)§ | 0.033 | 0.393 | |
| False discovery rate (q-value) | 13% | >50% | |
| TLR4 | |||
| TLR4-A | 27/29/26 | Referent | Referent |
| TLR4-B | 22/21/21 | 0.89 (0.74–1.07) | 0.94 (0.72–1.24) |
| TLR4-C | 14/13/15 | 0.86 (0.67–1.09) | 1.08 (0.79–1.49) |
| TLR4-D | 7/5/6 | 0.60 (0.44–0.82) | 0.95 (0.60–1.48) |
| TLR4-E | 6/6/9 | 0.82 (0.61–1.10) | 1.36 (0.90–2.06) |
| TLR4-F | 5/7/5 | 1.15 (0.82–1.62) | 0.98 (0.62–1.54) |
| TLR4-Others | 18/19/19 | 0.67 (0.48–0.93) | 0.88 (0.54–1.43) |
| Global Wald test (p-value)§ | 0.008 | 0.788 | |
| False discovery rate (q-value) | 6% | >50% |
Probability-weighed haplotype frequencies among controls/myocardial infarction (MI) cases/ischemic stroke (IS) cases.
Odds ratios (95%CI) of logistic additive models comparing each haplotype to the referent haplotype adjusted for matching factors (age, sex, race, index year and hypertension status).
Global Wald test p-value for overall association of gene with disease.
In logic regression evaluation of associations of MI and ischemic stroke, separately, with combinations of all 34 SNPs of the 4 genes that were considered, signal test p-values were greater than 0.10 suggesting absence of sufficient evidence to support interactions between evaluated SNPs in MI or ischemic stroke. Results of our sensitivity analyses (by restricting participants to only whites and by combining both case groups) were similar to results already described. We also did not find significant effect modification of associations by gender, diabetes status, smoking, obesity and alcohol intake beyond what would have been expected by chance.
Discussion
We demonstrated associations of global PPARA and TLR4 genetic variations with MI risk. The PPARA rs4253623 SNP was associated with increased MI risk while the TLR4 rs1927914 SNP was associated with decreased MI risk. PPARA and TLR4 genetic variations were not associated with ischemic stroke risk. Global CETP and IL8 genetic variations were not associated with either MI or ischemic stroke. No significant interaction between SNPs of the 4 genes (CETP, IL8, PPARA and TLR4) in MI or IS was apparent in our study.
Investigators have previously reported associations between PPARA and TLR4 gene variations with cardiovascular diseases. Two PPARA SNPs, one in exon 5 (rs1800206) and another in intron 7 (rs4253778 in partial allelic association with rs1800206) were associated with risk of non-fatal MI.7 We did not directly genotype a non-synonymous PPARA SNP or the intron SNPs (rs4253778 or rs1800206). The PPARA SNP, rs4253765, in linkage disequilibrium with SNP rs4253778, was not associated with either MI or ischemic stroke in our study. The PPARA SNP, rs4253623, associated with MI risk in the current study has not been described before. The TLR4 non-synonymous SNP, rs4986790, has been associated with a 37–61% decrease in risk of acute coronary events such as MI in several case control studies and a meta analysis,16–18 though several other studies reported no associations.8,19–20 Similarly, we did not find a reduced risk of MI associated with that particular SNP in our study. Lin et al found an association of a SNP in intron 1 of the TLR4 gene (A119C), with risk of IS in an Asian study population.21 We did not genotype the A119C SNP directly or a SNP in linkage disequilibrium with this SNP. The intron 1 SNP, rs1927911, part of the TLR4-D haplotype associated with lower risk of MI in our study or the SNPs tagged by it have not been previously described.
Reports of associations between CETP variations and cardiovascular diseases are inconsistent.9–10, 22–24 The B allele of the CETP/TaqIB polymorphism (rs708272) was associated with reduced CHD risk in some,9, 22 but not other studies.23 Two tag-SNPs (rs17231506 and rs1800775) in linkage disequilibrium with the rs708272 SNP were not associated with risk of MI in our study. The –A allele of the CETP gene C629A SNP in the promoter region (in linkage disequilibrium with a non-synonymous SNP rs5882 and SNP rs708272) was associated with decreased cardiovascular death.24 The assay for rs289716 SNP, in significant LD with rs5882 in our panel, failed and thus we did not genotype that SNP in our population. Thompson et al investigated 9 CETP SNPs that span the whole gene and found that only SNP rs1800774 was associated with history of MI.10 We did not replicate that finding. The marginal global CETP gene and MI association in our study is potentially due to the marginal higher risk associated with having a copy of an uncommon haplotype. The 2 CETP SNPs, rs708273 and rs12720922, associated with ischemic stroke in our secondary analysis were not described before. Further, there was no global CETP gene and IS association weakening the evidence for the associations.
Although IL8 chemokine has been associated with a number of overlapping functions in inflammation mediation and growth promotion in vascular cells known to be involved in cardiovascular risk development3 and the protein has been associated with increased risk of cardiovascular disease such as unstable angina and acute MI,25–26 IL8 haplotypes and/or SNPs were not associated with risk of MI or ischemic stroke in our study. No previous study has investigated IL8 SNPs or haplotypes in relation to MI or stroke.
Potential reasons for inconsistencies between specific SNP-disease association findings reported in our study compared with others include differences in SNP minor allele frequencies, linkage disequilibrium structure and disease status (fatal/non-fatal) among study populations. Genetic variants associated with fatal events or short-term mortality would be under-represented in our case-control study. Study power may be an issue in our interaction analysis where we adjusted for multiple testing. Most of our study participants were white and our results may not be generalizable to other racial groups. Finally, since we did not measure biomarkers in the current study, further studies that investigate associations between genetic variations, counterpart proteins, intermediates and disease status are needed to clarify mechanisms of associations. In conclusion, in this population-based case control study, we reported associations of PPARA and TLR4 genetic variation with risk of incident non-fatal MI. We also identified novel specific SNP-disease associations that need further investigations.
Acknowledgment
This study was supported by the following grants from the National Health Lung and BloodInstitute: HL73410, HL60739, HL68639, HL43201, HL74745, HL68986 and 1-T32-HL07902.
The authors are indebted to the participants of the Heart and Vascular Health Studies for their cooperation. They are also grateful for the expertise provided by staff of the Cardiovascular Health Research Unit at the University of Washington.
Footnotes
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