Variants of the Interferon Regulatory Factor 5 Gene Regulate Expression of IRF5 mRNA in Atherosclerotic Tissue But Are Not Associated With Myocardial Infarction
Background— Signaling events after activation of toll-like receptors (TLRs) are important mechanisms promoting inflammation in the atherosclerotic plaque. INF regulatory factor 5 (IRF5) is one of the mediators of downstream effects of TLRs. Several single nucleotide polymorphisms (SNPs) in the IRF5 gene have been found to be associated with systemic lupus erythematosus.
Methods and Results— We examined IRF5 mRNA expression in carotid atherosclerotic tissue (n=99) and the case-control association between SNPs in the IRF5 gene with myocardial infarction (MI) (n=376+387) and unstable coronary artery disease (CAD) (n=3101+445). Among unstable CAD patients, association of IRF5 SNPs with recurrent coronary events (n=401) was also investigated. The IRF5 mRNA expression was increased in atherosclerotic tissue compared with control tissue (P<0.001). Significant associations with IRF5 expression was observed for 6 of 10 SNPs in the study. However, the IRF5 SNPs examined were neither associated with the risk of precocious MI, nor with unstable CAD or risk of recurrent cardiovascular events in unstable CAD patients.
Conclusions— IRF5 mRNA is expressed in cells in atherosclerotic tissue and its expression is modified by SNPs in the IRF5 gene. Genetic variation at the IRF5 locus was, however, not associated with CAD or related phenotypes.
- acute coronary syndromes
- gene regulation
- genetics of cardiovascular disease
- arterial thrombosis
Coronary artery disease (CAD), a clinical manifestation of atherosclerosis, is one of the leading causes of death and morbidity worldwide.1 The understanding of the atherosclerotic process has increased through the demonstration of infiltration of inflammatory cells such as T-cells and macrophages into the atherosclerotic plaque and the recognition of cytokine, chemokine, and growth factor production by these cells.2 In addition, systemic proinflammatory cytokines are elevated in healthy individuals at increased risk of developing CAD and in CAD patients who suffer recurrent coronary events.3,4 Polymorphisms in genes encoding components of the inflammatory system have also been associated with CAD.5,6
The biological mechanisms that promote inflammation in atherosclerotic lesions are less well known. However, upregulation of pattern recognition receptors, a mechanism which coincides with the infiltration of monocytes through the activated endothelium, seems to be a crucial step for the initiation of chronic inflammation.7,8 The toll-like receptors (TLRs) constitute a family of pattern recognition molecules, which has a distinct role in cell activation that leads to inflammatory responses.8–10 TLRs specifically recognize pathogen associated molecules and are the master regulators of downstream immune responses. These events are thought to be mainly mediated via activation of the NFκB and MAPK pathways. A third pathway that is increasingly recognized in the TLR-mediated immune response involves the INF regulatory factor family, which seems to be essential for the regulation of type-1 INF expression.11 One member of this family, INF regulatory factor 5 (IRF5), has a critical function in downstream signaling of TLRs and the subsequent production of the cytokines interleukin (IL)-6, tumor necrosis factor (TNF)-α, and IL-12.12 Polymorphisms in the IRF5 gene have been associated with systemic lupus erythematosus (SLE),13 rheumatoid arthritis (RA),14 inflammatory bowel disease (IBD),15 and multiple sclerosis (MS).15a It has been demonstrated that mice deficient in IRF5 are hyporesponsive to lipopolysaccharide (LPS) exposure compared with wild-type mice.12 LPS is recognized by TLR-4 and, thus, it seems that IRF5 also mediates downstream effects of TLR-4 ligation. Polymorphisms in the TLR-4 gene have been associated with CAD, and it has been shown that subclinical endotoxemia is indicative of enhanced atherosclerotic progression in a prospective study of healthy individuals.16–19 Given the suggested role of IRF5 in TLR4 signaling, we hypothesized that IRF5 could be involved in atherosclerosis and CAD, and we used molecular genetic methodologies to address this hypothesis. The mRNA expression levels of IRF5 in carotid atherosclerotic tissue and in normal tissue were determined and compared between the types of tissue and genotypes of single nucleotide polymorphisms (SNPs) in the IRF5 gene. Association of IRF5 polymorphisms with risks of premature myocardial infarction (MI), unstable CAD, and recurrent coronary events in patients with unstable CAD were examined in 3 well characterized patient cohorts. In addition, associations between genetic variants in the IRF5 gene and CAD severity, ascertained by quantitative coronary angiography, and plasma concentrations of CRP and IL-6 were investigated.
Atherosclerotic Plaque Tissue
A total of 99 specimens of human plaque tissue were obtained from the Biobank of Karolinska Endarterectomies study (BIKE).20 BIKE includes samples of atherosclerotic tissue collected from patients undergoing carotid endarterectomy in the Department of Vascular Surgery at the Karolinska University Hospital. The median age among patients was 72 (36, range) and the mean body mass index was 25.4 (3.1, standard deviation). In patients, 69 individuals were men and 30 were women. Thirty percent of the patients were present smokers. Tissue specimens from patients were advanced lesions (Stary Class V-VIII), with or without macroscopic signs of plaque rupture and superimposed thrombosis. Control vessels were obtained from the iliac arteries of organ donors without macro- or microscopic signs of atherosclerotic disease (n=8).
Subjects in the Study of IRF5 Polymorphisms in Relation to Risk of Premature MI
The biobank and database of the Stockholm Coronary Atherosclerosis Risk Factor study (SCARF)21 was used for the first genotype-phenotype association study. The SCARF study enrolled 387 survivors of a first MI before the age of 60, in whom DNA and plasma samples were obtained 3 months after the index event. Coronary angiograms (n=243) obtained routinely in 2 of the 3 participating hospitals were analyzed by quantitative coronary angiography (QCA) using the Medis QCA-CMS system, as described.21 For each patient, a control person matched by age, gender and residence area was recruited from the population registry. The methods for determining plasma concentrations of CRP and IL-6 have been published.21
Subjects in the Studies of IRF5 Polymorphisms in Relation to Risk of Unstable CAD and Risk of Recurrent Events in Patients Presenting With Unstable CAD
The Scandinavian multicenter study FRagmin and fast revascularization during InStability in Coronary artery disease-II (FRISC-II) enrolled 3489 patients with unstable angina or non-ST elevation MI.22 The patients were followed regarding death and MI for 12 months by an independent end point committee. The primary end point investigated in the present study was the composite of cardiovascular death and fatal or nonfatal MI (n=401 events). A control group of apparently healthy individuals (n=445) with similar age and gender proportions was recruited from the Swedish population registry. Only individuals without a clinical history of cardiovascular disease or cardiovascular risk factors and with a normal electrocardiography and normal routine blood chemistry were included.23 The methods for quantification of CRP and IL-6 in the FRISC-II study are described elsewhere.24,25
Informed consent was obtained from all subjects recruited to the 3 patient cohorts. The investigations were approved by either the Ethical Committee of Northern Stockholm (BIKE and SCARF) or the Ethical Committee at Uppsala University (FRISC-II and FRISC-II controls). The study protocols were in agreement with institutional guidelines and the principles set forth in the Declaration of Helsinki.
Polymorphism Selection and Genotyping
The polymorphisms selected for the study included SNPs for which association with either SLE,13,26,27 RA,14 or IRF5 mRNA26–29 expression have been reported. The positions of selected SNPs are shown in Figure 1. The SNP rs2004640 is located at the splice junction of the alternative exon 1B of IRF5 where it alters a consensus splice donor site, which allows expression of alternative IRF5 mRNA transcripts containing exon 1B.28 Association with IRF5 mRNA expression levels has been reported for the SNPs rs2280714 and rs10954213.26,29
The SNPs were genotyped at the SNP technology platform in Uppsala (www.genotyping.se) by multiplex, fluorescent single-base extension using the SNPstream system (Beckman Coulter), with the exception of SNP rs4728142 which was typed by homogeneous fluorescent single-base extension with detection by fluorescence polarization (Analyst AD, Molecular Probes). The sequences of the primers used in the genotyping assays are available in supplemental Table I (available online at http://atvb.ahajournals.org). The genotype call rate was on average 96.9% and the accuracy, as estimated from 553 genotype comparisons between repeated assays (13% of the genotypes), was 99.94%. All genotype distributions conformed to Hardy-Weinberg equilibrium (Fisher’s exact test, P>0.01).
IRF5 Expression Analysis in Carotid Plaques
Total RNA was isolated from atherosclerotic lesions using the RNA FastRNA Kit, Green (Qbiogene), and stored at −70°C until cRNA synthesis. The integrity, quantity, and quality of the RNA-samples were tested using the Agilent BioAnalyzer. Ten micrograms of total RNA was used to generate biotin labeled cRNA. The reaction was performed according to the standard Affymetrix protocol, whereupon it was hybridized to Affymetrix HG_U133 A (or HG_U133A plus 2.0) Genechip. Probe level intensity data were analyzed in the GeneSpring analysis software (Silicon Genetics). The procedure and analyses have been described in detail elsewhere.20
IRF5 mRNA levels were determined in 70 individuals; 38 patients and 5 control tissue specimens were analyzed using the HG_U133 A chip, and 27 patient tissue specimens were analyzed using the U133A plus 2.0 chip. We used a quantitative polymerase chain reaction (PCR) (qPCR) Taqman assay to evaluate the data acquired by the Affymetrix Genechip (Applied Biosystems predesigned, Hs00158114_m1, exon 2 to 3 boundary). IRF5 expression levels were normalized against the expression of cyclophilin-A, as control for cDNA quality and quantity. A total number of 99 patients and 8 controls were available for the qPCR measurements.
Two IRF5 probe sets were available in both versions of the Affymetrix Genechip arrays; the probes 205468_s_at and 205469_s_at. The 205468_s_at probe hybridizes to exons 1 to 4 and the 205469_s_at to the 3′UTR region. The expression levels determined using the probes 205468_s_at and 205469_s_at were correlated (Spearman R2=0.56). However, the signals from probe 205469_s_at were stronger and more dynamic in comparison with the signals from probe 205468_s_at. Therefore, only 205469_s_at was used in the association analyses. The average signal intensity for both probe-sets was higher in U133A plus 2.0 compared with U133A. Thus, we were discouraged to merge the expression data into one data set and the data sets were analyzed independently.
The Student t test was performed on log-transformed IRF5 mRNA expression values to test for differences in expression levels between atherosclerotic- and control tissue specimens. To compare IRF5 mRNA expression levels between IRF5 genotypes, we performed linear regression analysis under an additive genetic model using the PLINK software (the DOS version).30 Comparisons of plasma concentrations of IL-6 and CRP between IRF5 genotypes were performed using analysis of variance. Calculations of LD and statistical tests for differences in allele- or haplotype frequencies between cases and controls were performed in Haploview 4.0.31 Multiple test correction was warranted for the 2 independent haplotype blocks across the IRF5 locus, given the strong LD. Accordingly, a probability value below 0.025 was considered statistically significant, which is consistent with a confidence level of 95% and correction for 2 independent tests. A combined probability value for the 4 independent studies of plasma markers in relation to IRF5 genotypes were calculated using Fisher’s combination procedure according to
with 2k degrees of freedom.32,33 The Kruskal-Wallis test was used in the comparison of QCA measures of CAD severity between IRF5 genotypes.
IRF5 mRNA Expression in Atherosclerotic Tissue
We compared the IRF5 expression level between atherosclerotic tissue and control tissue, and IRF5 expression in relation to IRF5 genotypes. The mean IRF5 mRNA expression level was significantly higher in atherosclerotic tissue from carotid endarterectomy specimens than in control tissue (Figure 2). In contrast, no association between IRF5 expression level and clinical symptoms, smoking status, body mass index, or medications were observed (data not shown). There was no difference in IRF5 expression level between male and female subjects in the study (0.89 and 0.93, respectively; P=0.66).
Polymorphisms in the IRF5 Gene and Risk of Precocious MI, Unstable CAD, and Recurrent Events in Patients Presenting With Unstable CAD
As detailed in a previous report,21 the postinfarction patients and controls in the SCARF study were carefully phenotyped and, as expected, differed in several established clinical and biochemical risk factors. The allele frequencies of the IRF5 polymorphisms investigated did, however, not differ between patients and controls (Table 3). Haplotype analysis based on all SNPs determined in the study did not alter this lack of association.
Evidence of strong gene-environment interactions in complex diseases has been provided in recent years. Patients in a young MI cohort such as those enrolled in the SCARF study generally display a lower prevalence of comorbidities, less extensive atherosclerosis, and a lower degree of inflammation compared with MI patients of older age. Accordingly, interactions between environmental factors and IRF5 may be undetectable in a cohort of young MI patients. Therefore, we investigated the possible association of IRF5 SNPs and haplotypes with unstable CAD and recurrent coronary events in a prospective study of patients with unstable CAD and matched controls. However, no associations of IRF5 SNPs or haplotypes with unstable CAD or recurrent cardiovascular events were observed (Table 3).
Systemic Inflammation and CAD Severity According to IRF5 Polymorphisms
To test whether IRF5 polymorphisms were associated with plasma concentrations of established biomarkers of inflammation such as IL-6 and CRP, we examined these genotype-phenotype associations in the 4 different samples of patients and controls (Table 4). Significant associations of IL-6 concentrations with rs3807306 and rs10954213 genotypes were observed in the FRISC-II controls (P=0.005 and P=0.02). However, these results were not consistent across all patient/control collections, and the combined probability value indicated no associations (P=0.08 and P=0.06, respectively).
The QCA analyses in the 243 patients with precocious MI undergoing coronary angiography evaluated minimum luminal diameter (MLD), mean segment diameter (MSD), percentage diameter stenosis, total plaque area, and the number of vessels with significant stenoses (<50%). Neither of these measures of CAD severity was associated with IRF5 genotype (data not shown).
We demonstrated that IRF5 mRNA is expressed in human carotid plaques and that IRF5 expression in atherosclerotic tissue is influenced by several SNPs within (and flanking) the IRF5 gene. However, despite the apparent allelic associations with IRF5 mRNA levels, the SNPs in our study were neither associated with precocious MI, nor with risk of unstable CAD or recurrent coronary events in patients presenting with unstable CAD.
The substantial difference between IRF5 mRNA expression levels in atherosclerotic tissue and control tissue may reflect that expression of IRF5 mRNA in a local and systemic state of chronic inflammation is enhanced. However, the composition of different cell-types is likely to vary between the patient plaques and the control tissue of iliac arteries, and thus the data should be interpreted with some caution.
Several of the IRF5 SNPs in the present study have previously been reported to influence IRF5 gene expression. First, the substitution of T to G at SNP rs2004640 changes a consensus splice site for an alternative exon 1, which introduces the transcription of a low-abundance IRF5 transcript containing exon 1b in human monocytes.34 Secondly, Graham et al reported from an investigation of IRF5 mRNA levels in EBV-transformed CEPH lymphocytes that, similar to our findings, AA homozygotes of the rs10954213 polymorphism express higher levels of IRF5 mRNA.26 The authors suggested that the A allele of rs10954213 creates a polyA+ signal site that causes expression of IRF5 mRNA with a shorter 3′UTR, which is more stable than the longer variant expressed from the G allele. Thirdly, our group has recently identified an insertion/deletion of the nucleotides CGGGG, which is positioned at 64 bp upstream of the first untranslated exon of IRF5. The longer risk allele of the CGGGG polymorphisms contains an additional SP1 binding site and seems to increase the expression of IRF5 transcripts containing exon 1a (Sigurdsson, Kristiansdottir). In the present study, the CGGGG is relatively well tagged by the SNP rs4728142, which has an R2 correlation of 0.86 with the CGGGG polymorphism. The presence of multiple functional SNPs in the IRF5 gene would explain the observation that several of the IRF5 SNPs in the present study were associated with IRF5 mRNA expression in cells in atherosclerotic tissue.
The statistical power of the present study argues against the possibility that the lack of association between IRF5 SNPs and MI/unstable CAD was attributable to a type-2 error. The following assumptions were stated in the process of designing our study: detection of a 1.4 or higher risk ratio, alpha <0.05, >90% power, and an additive genetic model. These assumptions were fulfilled for all but the rs12539741 SNP, which had the lowest minor allele frequency of the SNPs examined in our study. In the FRISC-II study, even a lower risk ratio at 1.3 would have been detectable with all SNPs except for the rs12539741. However, more modest effects of IRF5 SNPs on the studied traits cannot be excluded.
A previous study reported that the LPS-induced IL-6 production was attenuated in IRF5-deficient mice.13 We investigated the relationship between IRF5 and cytokine production, by assessing the association between plasma concentrations of IL-6 and CRP and the IRF5 SNPs related with IRF5 expression levels. The lack of consistent association between IRF5 genotypes and systemic CRP and IL-6 concentrations converges with our findings suggesting that genetic polymorphisms in the IRF5 gene do not confer susceptibility to MI or unstable CAD. Similarly, the severity of CAD, as assessed by QCA in a representative selection of 243 patients with precocious MI, did not differ between patients with different genotypes of IRF5 polymorphisms related with splicing and mRNA expression. Based on conclusions drawn from the analyses of these CAD related phenotypes, our data suggest that differential IRF5 expression neither promotes atherosclerosis nor contributes to enhanced systemic inflammation during the acute coronary syndrome. Still, because expression of IRF5 was not measured in MI patients and because genetic regulation in patients with carotid atherosclerosis may differ from that in patients with coronary disease, extrapolation of the results obtained in patients with carotid atherosclerosis should be done with caution. Furthermore, the impact of allele-specific mRNA expression on mRNA translation into functional IRF5 protein remains unknown, and this also pertains to the effect on the putative functions of IRF5 protein in the downstream signaling pathway of TLR4.
Chronic inflammatory disorders such as SLE, RA, IBD, CAD, and MS are expected to, at least to some degree, share common disease mechanisms and genetic risk factors. An example of a common genetic risk factor is the promoter SNP rs3087456 in the MHC2TA gene, which was related to the risk of MI, MS, and RA.35 Since the first identification of IRF5 as a susceptibility gene for SLE, the results have been replicated in subsequent larger case-control studies26–28,36 and extended to include also risk of other inflammatory disorders, ie, RA, IBD,14,15 and MS.15a In contrast, we found no association between IRF5 SNPs and any of the cardiovascular disease traits investigated in the present study. These results may imply a less pronounced role of downstream IRF5 effects in CAD pathogenesis and that other mechanisms after TLR ligation, such as the ones mediated by other members of the IRF family, may be more active in subsequent inflammatory reactions. Alternatively, our results may argue against a strong role of type-1 INF expression in atherogenesis.
In conclusion, we found that the IRF5 gene transcript is present in carotid atherosclerotic tissue and that the IRF5 mRNA expression is influenced by several SNPs across the IRF5 locus. Polymorphisms in the IRF5 gene are, however, not associated with susceptibility to MI or unstable CAD. These findings indicate that the observed effects of IRF5 SNPs on IRF5 gene expression are either too small to influence the disease traits investigated or that IRF5 expression is not a casual factor in CAD development and progression.
We gratefully acknowledge the contributions of the BIKE, FRISC-II, and SCARF investigators.
Sources of Funding
This study was supported by grants from the Swedish Research Council (AS-11568, AH-8691, ACS-14436), the Swedish Heart-Lung Foundation, the K&A Wallenberg Foundation, the European Commission (LSHM-CT-2007–037273), the Stockholm County Council Project (560183), the Nanna Svartz Foundation, and the Magnus Bergvall Foundation. Genotyping was supported by the K&A Wallenberg Foundation via the Wallenberg Consortium North (WCN).
Original received October 9, 2007; final version accepted February 25, 2008.
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