Interferon regulatory factor 5 genetic variants are associated with cardiovascular disease in patients with rheumatoid arthritis

Introduction Rheumatoid arthritis (RA) is a complex polygenic inflammatory disease associated with accelerated atherosclerosis and increased cardiovascular (CV) disease risk. Interferon regulatory factor 5 (IRF5) is a regulator of type I interferon induction. Recently, researchers have described an association between multiple single-nucleotide polymorphisms of the IRF5 gene and some rheumatic disorders. In this study, we aimed to evaluate whether three different haplotype blocks within the IRF5 locus which have been shown to alter the protein function are involved in the risk of CV events occurring in Spanish RA patients. Methods Three IRF5 polymorphisms (rs2004640, rs2070197 and rs10954213) representative of each haplotype group were genotyped by performing TaqMan assays using a 7900HT Fast Real-Time PCR System with tissue from a total of 2,137 Spanish patients diagnosed with RA. Among them, 390 (18.2%) had experienced CV events. The relationship of IRF5 genotypes and haplotypes to CV events was tested using Cox regression. Results Male sex, age at RA diagnosis and most traditional risk factors (hypertension, dyslipidemia and smoking habit) were associated with increased risk for CV events in the RA population. Interestingly, a protective effect of both IRF5 rs2004640 GG and IRF5 rs10954213 GG genotypes against the risk for CV events after adjusting the results for sex, age at RA diagnosis and traditional CV disease risk factors was observed (hazard ratio (HR) = 0.6, 95% confidence interval (CI) = 0.38 to 0.92, P = 0.02; and HR = 0.58, 95% CI = 0.36 to 0.95, P = 0.03, respectively). Moreover, we detected a protective effect of the GTG haplotype against the risk for CV events after adjusting the results for potential confounding factors (HR = 0.72, 95% CI = 0.56 to 0.93, P = 0.012). Conclusions Our results reveal that IRF5 gene variants are associated with risk of CV events in patients with RA.


Introduction
Rheumatoid arthritis (RA) is a chronic inflammatory rheumatic disease associated with an increased risk for cardiovascular (CV) events and CV disease-related deaths compared to the general population [1]. Because of that, adequate stratification of CV disease risk has special relevance in RA patients. Researchers in several studies have demonstrated that RA is an independent risk factor for premature heart disease [2]. This process is the result of a combined effect of traditional CV disease risk factors [3], the magnitude and severity of a chronic inflammatory response [4] and genetic factors located inside [4] and outside the human leukocyte antigen (HLA) region [5][6][7][8].
Type I interferons (IFNs) are signaling molecules involved in both innate and adaptive immunity. Misregulated expression of type I IFN genes has been observed in peripheral white blood cells of patients with several autoimmune diseases [9]. In this regard, interferon regulatory factor 5 (IRF5) has been involved in the regulation of type I IFN gene transcription [10,11]. This protein is critical for the production of proinflammatory cytokines [12], such as TNF-α, interleukin 12 (IL-12) and IL-6, following Toll-like receptor signaling. IRF5 also acts as a molecular switch that controls inflammatory mechanisms mediated by macrophage cells [12]. IRF5 protein is encoded by the IRF5 gene, which is located in chromosome 7q32.1. Several genetic studies have described the relevance of multiple single-nucleotide polymorphisms (SNPs) of the IRF5 gene in different rheumatic disorders, such as RA and lupus erythematosus [13,14]. In this context, three groups of correlated IRF5 variants, designated as groups 1, 2 and 3, have been found to be independently associated with these inflammatory disorders and with different functional roles. Group 1 includes SNPs tagging a 30-bp in-frame insertion/deletion variant of exon 6 that alters protein stability. The association of group 2 SNPs seems to be explained by the T allele of IRF5 rs2004640 polymorphism that allows the expression of an alternative isoform and is associated with significantly higher levels of IRF5 expression. The association of group 3 SNPs is probably due to the rs10954213 A allele that creates an early polyadenylation site which leads to higher IRF5 expression [14].
Taking into account all of these considerations together in the present study, we analyzed the potential role of one tagging SNP of each group of independent signals (rs2004640, rs2070197 and rs10954213) in the risk for CV disease in a large and well-characterized cohort of patients with RA.  [15,16]. In all the cases, patients were assessed for the IRF5 rs2004640, rs2070197 and rs10954213 polymorphisms.

Patients and study protocol
Data on the main demographic data, clinical characteristics, CV disease risk factors and CV events of patients enrolled in the study are shown in Table 1. Three hundred ninety (18.2%) of the patients had experienced CV events. Definitions of CV events and traditional CV disease risk factors were established as previously described [4,17].

Genotyping
Patient DNA was obtained from peripheral blood using standard methods. The IRF5 rs2004640, rs2070197 and rs10954213 polymorphisms were genotyped with predesigned TaqMan SNP genotyping assays in a 7900HT Fast Real-Time PCR System according to the conditions recommended by the manufacturer (Applied Biosystems, Foster City, CA, USA). Negative controls and duplicate samples were included to check the accuracy of genotyping.

Statistical analysis
The genotype data were checked for deviation from Hardy-Weinberg equilibrium (HWE) using a previously described method [18]. The relationship of genotypes, alleles and haplotypes to CV events that occurred in the follow-up was tested using Cox regression adjusted for sex, age at RA diagnosis and traditional CV disease risk factors.
For that purpose, we used the most frequent genotype, allele and haplotype as reference variables. The end of follow-up was the first date of occurrence of the following: end of the study period, date of death or date of CV event. Follow-up time was estimated as the difference between the RA diagnosis date and the end of follow-up. Patients without CV events during the follow-up time and those who died by any non-CV-event-related cause were censored. The results are expressed as hazard ratios (HRs) with 95% confidence intervals (CIs). In order to have a reference population, 10,000 replications were generated by assigning CV events in randomized fashion to the actual studied population. The proportion of the risk for CV disease due to age at disease diagnosis, smoking history, hypertension and genetic variants at the IRF5 locus among the RA patients was estimated as the Nelson-Aalen cumulative HR. Comparative values between the different genetic models of inheritance for IRF5 polymorphisms were estimated using the Akaike Information Criterion (AIC). Statistical significance was defined as P ≤ 0.05, and all analyses were performed using Stata SE/12 statistical software (Stata Corp, College Station, TX, USA).

Results
The IRF5 rs2004640, rs2070197 and rs10954213 polymorphism genotype distributions were in Hardy-Weinberg equilibrium. The genotyping success rate was greater than 98% in all cases. Genotype and allele frequencies of the IRF5 rs2004640, rs2070197 and rs10954213 polymorphisms were in agreement with the data in the HapMap database [19]. Table 2 describes those factors that were associated with the risk of CV disease in our cohort of RA patients. As expected, sex and most of the traditional CV disease risk factors (hypertension, dyslipidemia and smoking habit) were associated with increased risk of CV events (P < 0.05 in all cases) ( Table 2). Interestingly, when we analyzed the IRF5 rs2004640, rs2070197 and rs10954213 polymorphisms separately, we observed a protective effect of the IRF5 rs2004640 GG genotype against the risk of CV events after adjusting the results for sex, age at RA diagnosis and traditional CV disease risk factors (hypertension, diabetes mellitus, dyslipidemia, obesity and smoking habit) (HR = 0.6, 95% CI = 0.38 to 0.92, P = 0.02) ( Table 2). Additionally, the risk of CV disease was decreased in the group of RA patients who carried the IRF5 rs10954213 GG genotype after we adjusted the results for potential confounders (HR = 0.58, 95% CI = 0.36 to 0.95, P = 0.03) ( Table 2). These results were also significant after we estimated by simulation (P = 0.034 and P = 0.049 for the IRF5 rs2004640 GG and rs10954213 GG genotypes, respectively) ( Table 2). However, we found no statistically significant differences when we assessed the IRF5 rs2070197 polymorphism according to the presence or absence of CV events (Table 2). In a further step, we combined the three polymorphisms to create haplotypes ( Table 3). As shown in Table 3, we detected a protective effect of the GTG haplotype after adjusting the results for sex, age at RA diagnosis and traditional CV disease risk factors (HR = 0.72, 95% CI = 0.56 to 0.93, P = 0.012).
Additional file 1: Table S1 describes the proportion of the CV disease risk disease for RA patients due to age at disease diagnosis, smoking history, hypertension and the genetic variants at the IRF5 locus. Additional file 2: Table  S2 displays the genetic model of inheritance of our analysis. Interestingly, as shown in Additional file 1: Table S1, the presence of GG genotypes in the IRF5 rs2004640 and rs1095213 polymorphisms decreased CV disease risk in 0.7% and 1% of patients, respectively (Additional file 1: Table S1). In addition, smoking, age at disease diagnosis and presence of hypertension increased the risk of CV disease (Additional file 1: Table S1). Further, as shown in Additional file 2: Table S2, the AIC estimation for the recessive model showed the lower level in the IRF5 rs2004640 and rs1095213 polymorphisms, which made it the preferable model for our study (Additional file 2: Table S2).

Discussion
CV disease is the most common cause of premature mortality in patients with RA [1,2]. The augmented CV disease mortality observed in this pathology is the result of a compound effect mediated by traditional CV disease risk factors and chronic inflammation [3,4]. Because genes have also been associated with an increased risk of CV disease in RA, research in the past several years has been focused on the search for genetic markers that may improve the identification of RA patients at risk of experiencing CV events [4][5][6][7][8].
Outside the HLA region, IFN pathway genes, which encode cytokines with critical modulatory effects on innate and adaptive immunity, have been shown to represent a key component of the genetic network leading to autoimmune processes. In this context, several polymorphisms in the IRF5 gene (a regulator of type I IFN induction) [10,11] are associated with an increased risk of immune-mediated diseases [13,14]. Because of that, in this study we analyzed three genetic variants (IRF5 rs2004640, rs2070197 and rs10954213) as representatives of three different IRF5 haplotype blocks [14]. To the best of our knowledge, our study constitutes the largest series of RA patients assessed for the potential influence of IRF5 polymorphisms in the risk of CV disease. Interestingly, when we studied each of the polymorphisms separately, our results revealed a protective effect of the IRF5 rs2004640 GG and IRF5 rs10954213 GG genotype variants against the risk of CV events in RA. Moreover, when we analyzed all the genetic variants together to create haplotypes, our results revealed a protective effect of the GTG haplotype (the one that carries both G alleles of IRF5 rs2004640 and rs10954213 polymorphisms) against the risk of CV disease. Because the protective effect of the mutant allele of the IRF5 rs2004640 and rs1095213 polymorphisms is seen only in homozygosis, we can conclude that our study is adjusted to a recessive model of inheritance. Additionally, the results derived from our study are in accordance with those obtained in other pathologies [20,21]. In this context, the results of a recent study of individuals diagnosed with RA revealed an association between the IRF5 rs2004640 polymorphism and subclinical atherosclerosis [22].
The results obtained in this study provide additional evidence on the potential role that genetic factors may play in the development of CV disease in RA. The search for genetic markers associated with CV disease in RA may be important to establishing a better characterization of RA patients at risk of CV disease. Improved understanding of these underlying genetic may be useful to establish future therapeutic targets to decrease the risk of CV disease in RA patients.

Conclusion
Our results reveal that IRF5 gene variants are associated with CV disease in RA patients.