Effect of Interleukin-27 Genetic Variants on Atrial Fibrillation Susceptibility

Abstract

Aim:

Atrial fibrillation (AF) is the most common cardiac arrhythmia. No data are available on the association between the polymorphisms of interleukin-27 (IL-27) and AF in the Chinese Han population. This study was performed to determine if polymorphisms within the IL-27 gene are involved in AF susceptibility.

Methods:

Two hundred seventy AF patients and 303 healthy individuals were examined for two IL-27 gene polymorphisms (rs153109 and rs17855750) by polymerase chain reaction-restriction fragment length polymorphism methodology.

Results:

An association between the IL-27 single nucleotide polymorphism (SNP) rs153109 and AF was found in Chinese Han population. The G allele and GG genotype of rs153109 were associated with increased AF risk (odds ratio [OR]: 1.35, 95% confidence intervals [CI] = 1.06-1.71, p = 0.02 and OR: 1.66, 95% CI = 1.03-2.65, p = 0.03 in the recessive genetic model, respectively). The significance of the association between the GG genotype and AF risk did not survive a Bonferroni's correction. Similarly, no significant differences in the allele and genotype frequencies of the rs17855750 SNP were observed between the AF patients and controls.

Conclusions:

Our findings indicated that the IL-27 genetic polymorphisms may be associated with susceptibility of AF in Chinese Han population.

Introduction

Atrial fibrillation (AF) is the most common cardiac arrhythmia, and it is characterized by disorganized atrial depolarization and disappearance of atrial contraction that result in the absence of P wave in electrocardiogram (ECG) and impaired cardiac function, which may or may not be symptomatic. It often increases the risk for heart failure, thromboembolic diseases such as cardioembolic stroke, and mortality, resulting in large public health costs. It has been demonstrated that there was a progressive increase in prevalence of AF between 1990 and 2010. The estimated number of individuals with AF globally in 2010 was 33.5 million (20.9 million men and 12.6 million women) (Chugh et al., 2014). Although beta-blockers, amiodarone, propafenone, and even radiofrequency ablation have been widely administrated for the patients with AF, the morbidity and mortality of AF are still very high. Because the elusive pathogenesis mechanisms of AF still remain unknown, our treatments are not targeted to specific etiologies. Better understanding of these underlying mechanisms can be expected to improve prevention and therapy of AF.

Interleukin (IL)-27, composed of Epstein-Barr virus-induced gene 3 and IL-27p28, is a heterodimeric cytokine and belongs to IL-12/IL-23 heterodimeric family of cytokines. With a mice gene knockout model, researchers have found that IL-27 is an inflammatory cytokine, which associates with proliferation and interferon-γ production in CD4+ T cells (Pflanz et al., 2002; Iwasaki et al., 2015). Accumulating data confirmed a suppressive role of IL-27 in experimental autoimmune encephalomyelitis by suppressing the development of IL-17-producing T cells and indicated that IL-27 may be a useful therapeutic agent for inhibiting autoimmune inflammation (Batten et al., 2006; Fitzgerald et al., 2007). Furthermore, using mice deficient in the IL-27 receptor, IL-27 has been found to negatively regulate ectopic lymphoid-like structure development in rheumatoid arthritis through control of effector T cells (Jones et al., 2015). These observations collectively suggest that both pro- and anti-inflammatory activities of IL-27 can play an important role in the different classes of inflammation, thereby contributing to the pathogenesis of AF.

Studies in inflammatory or autoimmune diseases have shown that two polymorphisms of IL-27 gene (rs153109 and rs17855750) might be the genetic marker for susceptibility of these diseases and would contribute to their etiology. Allergic rhinitis (AR) is an allergic inflammation of the nasal airways. For rs153109 of IL-27 gene, TT genotype and the T allele were increased significantly compared with the controls. Meanwhile, for rs17855750 of IL-27 gene, the TT genotype and T allele were associated with a significantly increased risk of AR (Shen et al., 2014). Wang et al. (2014) further confirmed the association between IL-27 gene polymorphisms and inflammatory diseases by showing there were significant differences between Crohn's disease patients and controls in allele distributions of rs153109. In one congenital heart defect study, these two polymorphisms of IL-27 gene have also been investigated as to whether they have an association with ventricular septal defect and atrial septal defect. It has been shown that the rs153109 of the IL-27 gene may be associated with the susceptibility to congenital heart defects. In this study, we evaluated the role of these two polymorphisms of IL-27 gene (rs153109 and rs17855750) in susceptibility to AF.

Materials and Methods

Study subjects

This study enrolled 270 nonfamilial AF patients from West China Hospital of Sichuan University and Affiliated Hospital of North Sichuan Medical College between September 2010 and December 2015. The recruitment criteria of this study were as follows: (1) the ECG or Holter records indicated AF and (2) they had no evidences of valvular disease, structural heart disease, cardiomyopathy, and coronary heart diseases. Patients who had previously received ablation were excluded. Among 270 AF patients, 195 patients underwent catheter ablation for refractory to antiarrhythmic drugs at our hospitals. Control subjects were 303 genetically unrelated individuals from a routine health survey. All patients and controls received echocardiographic evaluation of left ventricle function and left atrial (LA) size. Informed consent was obtained from participants. Study protocols were approved by the hospitals' ethics committees.

Determination of genotypes

Genomic DNA of each individual was isolated from ethylenediaminetetraacetic acid-anticoagulated peripheral vein blood using a DNA Isolation Kit from BioTeke (BioTeke Corporation, Beijing, China), and the procedure was performed according to instruction manual. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method was used to genotype these two single nucleotide polymorphisms (SNPs) of IL-27 gene. PCR-RFLP was carried out as follows: 50 ng of genomic DNA was amplified in a total volume of 25 μL reaction mixture containing 75 mM Tris-HCl (pH 9), 1.5 mM MgCl₂, 150 mM KCl, 2 mM (NH₄)₂SO₄, 200 pmol dNTP, 10 pmol of each primer, and 1.5 U of Taq DNA polymerase. PCR conditions were as follows: 94°C for 5 min, 36 cycles at 94°C for 30 s, 30 s at 64°C for rs153109, 66°C for rs17855750, and 72°C for 55 s and a final elongation at 72°C for 10 min. PCR products were digested with corresponding restriction enzyme for 2 h and analyzed by 6% polyacrylamide gels with silver staining. The primers and restriction enzymes used in the genotyping analysis have been reported previously (Zhou et al., 2015). The PCR primers for rs153109 were 5′-CTGATCCTGACCTCACTCAACGC-3′ (forward) and 5′-CTGACTGGGACTGGGACTCAGC-3′ (reverse). PCR products of 119 bp were digested with BstUI. Allele G yields two fragments of 22 and 97 bp, and allele A yields a 119 bp band (Supplementary Fig. S1A; Supplementary Data are available online at www.liebertpub.com/gtmb). The PCR primers for rs17855750 were 5′-ATCTCGCCAGGAAGCTGCGC-3′ (forward) and 5′-CTGTTAGTGGGGGCCAGAAGGGA-3′ (reverse). PCR products of 120 bp were digested with BstUI. Allele G yields two fragments of 19 and 101 bp, and allele T yields a 120 bp band (Supplementary Fig. S1B). About 10% of the samples were confirmed by direct sequencing.

Statistical analysis

Data analyses were carried out using SPSS 12.0 statistical software (SPSS, Inc., Chicago, IL).

In univariate analysis, Student's t-test was performed for quantitative variables and Pearson's chi-squared test for categorical variables. Allelic and genotype frequencies were obtained by directed counting. Hardy-Weinberg equilibrium was tested by the chi-squared test and performed, respectively, on cases and controls. Moreover, SNPstats has been used to calculate genotypic association test in a case-control pattern, assuming codominant, dominant, recessive, or overdominant genetic models (Sole et al., 2006). The genotype frequencies and the allele frequencies in the cases and control groups were compared by the Pearson chi-squared test. Odds ratio (OR) and respective 95% confidence intervals (CI) were reported to evaluate the effects of any difference in allelic and genotype distribution. Bonferroni corrected p-value of 0.025 was set for a type 1 error. When the p-value was <0.025, the differences of allele and genotype distribution were regarded as statistically significant in AF patients compared to control subjects. The study power was calculated using the QUANTO program (http://biostats.usc.edu/index.html). All tests were two tailed.

Results

The general characteristics and echocardiographic parameters of the study population are summarized in Table 1. Gender and age between cases and controls were compared with Pearson's chi-squared test and Student's t-test, respectively, and significant differences were not detected (Table 1). As expected, the AF group had a larger LA (38.79 vs. 33.05 mm, p < 0.05), similar left ventricle ejection fraction (62.28% vs. 63.99%, p > 0.05), compared with the control group. Furthermore, the AF group had a higher prevalence of New York Heart Association (NYHA) class II, no subject of NYHA class III has been found in the control group, indicating that AF patients have more severe NYHA functional class.

Table 1.

Comparisons of Characteristics of Controls and Atrial Fibrillation Patient

	Control (n = 303)	AF (n = 270)
Gender (male/female)	183/120	180/90
Age (years)	59.12 ± 7.09	58.10 ± 7.11
PAF (%)	0	71.1
NYHA	I: 240, II: 63	I: 82 II: 133 III: 55
LVEF (%)	63.99 ± 4.93	62.28 ± 4.65
LA (mm)	33.05 ± 1.94	38.79 ± 3.48^*

Data are presented as the mean value ± SD or number (%) of subjects.

p < 0.05 compared between controls and AF patient.

PAF, paroxysmal atrial fibrillation; NYHA, New York Heart Association; LVEF, left ventricular ejection fraction; LA, left atrium; SD, standard deviation; AF, atrial fibrillation.

All 573 subjects were successfully genotyped. There was no deviation from Hardy-Weinberg equilibrium in control groups (p > 0.05), indicating that the frequencies fell into the expected equilibrium and were thus randomly distributed. Using chi-squared test, we compared genotype and allele frequencies between cases and controls. Nominally, the G allele and GG genotype of rs153109 have been found to be associated with increased AF risk (OR: 1.35, 95% CI = 1.06-1.71, p = 0.02 and OR: 1.66, 95% CI = 1.03-2.65, p = 0.03 in the recessive genetic model, respectively). We found an association of risk allele G of rs153109 with AF risk, and an association of GG genotype of rs153109 with AF risk did not withstand Bonferroni's correction (corrected p-value = 0.025). No significant difference of SNP rs17855750 in the allele and genotype frequencies was observed between the AF patients and controls (Table 2). The allele and genotype frequencies of these two SNPs between AF patients and controls and the statistical analysis results are shown in Table 2. At the 0.05 level of significance with the two-sided test for these two polymorphisms, our study had 91.6% and 84.1% power (for rs153109 and rs17855750 polymorphisms) to detect an effect with a relative risk of 1.8 in the group of AF patients and healthy controls.

Table 2.

Distribution of Single Nucleotide Polymorphism Genotype in Interleukin-27 Between Cases and Controls and Their Association with Atrial Fibrillation Risk

Model	Genotype	Controls n = 303	Cases n = 270	OR (95% CI)	p-Value
rs153109
Codominant	AA	131 (43.2%)	96 (35.6%)	1.00
	AG	137 (45.2%)	126 (46.7%)	1.26 (0.88-1.79)	0.05
	GG	35 (11.6%)	48 (17.8%)	1.87 (1.12-3.11)
Dominant	AA	131 (43.2%)	96 (35.6%)	1.00	0.06
	AG/GG	172 (56.8%)	174 (64.4%)	1.38 (0.99-1.93)
Recessive	AA/AG	268 (88.5%)	222 (82.2%)	1.00	0.03
	GG	35 (11.6%)	48 (17.8%)	1.66 (1.03-2.65)
Overdominant	AA/GG	166 (54.8%)	144 (53.3%)	1.00	0.73
	AG	137 (45.2%)	126 (46.7%)	1.06 (0.76-1.47)
Allele	A	399 (65.8%)	318 (58.9%)	1.35 (1.06-1.71)	0.02
	G	207 (34.2%)	222 (41.1%)
rs17855750
Codominant	TT	252 (83.2%)	233 (86.3%)	1.00
	TG	45 (14.8%)	30 (11.1%)	0.72 (0.44-1.18)	0.38
	GG	6 (2%)	7 (2.6%)	1.26 (0.42-3.81)
Dominant	TT	252 (83.2%)	233 (86.3%)	1.00	0.30
	TG/GG	51 (16.8%)	37 (13.7%)	0.78 (0.50-1.24)
Recessive	TT/TG	297 (98%)	263 (97.4%)	1.00	0.62
	GG	6 (2%)	7 (2.6%)	1.32 (0.44-3.97)
Overdominant	TT/GG	258 (85.2%)	240 (88.9%)	1.00	0.18
	TG	45 (14.8%)	30 (11.1%)	0.72 (0.44-1.17)
Allele	T	549 (90.6%)	496 (91.9%)
	G	57 (9.4%)	44 (8.1%)	1.17 (0.78-1.77)	0.45

Significant p-values (<0.025) are in bold face.

OR, odds ratio; CI, confidence intervals.

Discussion

The association of IL-27 polymorphism is determined in several autoimmune and inflammatory diseases, including ulcerative colitis, Crohn's disease, allergic rhinitis, and rheumatoid arthritis (Paradowska-Gorycka et al., 2014; Shen et al., 2014; Wang et al., 2014; Yamamoto-Furusho et al., 2016). In our study, we found that the G allele of rs153109 was significantly higher in AF patients than in controls. It has suggested that G allele may be a risk factor for AF in Chinese Han population compared with the A allele.

Recently, the inflammation has been highlighted in the pathogenesis of AF. First, it has been demonstrated that inflammation might play a significant role in the initiation, maintenance, and perpetuation of AF, and elevated IL-6 and C-reactive were observed in peripheral blood of a subset of AF patients (Issac et al., 2007). In addition, using a community based cohort, concentrations of tumor necrosis factor (TNF)-α, TNF-α soluble receptor (SR) I, and TNF-a SR II had been found to be associated with a greater adjusted risk of incident AF (Dewland et al., 2015). Furthermore, AF was also accompanied by atrial fibrosis, which was linked to the inflammation of atrial myocardium. A human anti-IL-1β monoclonal antibody, designed to reduce inflammation, could be promising therapeutic opportunity for AF patients (Ferrari et al., 2016).

C-reactive protein (CRP) is a marker of inflammation in several cardiovascular diseases; recent studies have uncovered that persistent AF patients had higher CRP level than paroxysmal AF patients, indicating that it may take part in initiation and persistence of AF (Chung et al., 2001). One meta-analysis further confirmed that increased baseline CRP levels were associated with higher risk of AF recurrence after successful electrical cardioversion, while AF patients with elevated CRP might be improved by the use of anti-inflammatory agents (Liu et al., 2007). Furthermore, Yamashita et al. (2010) have investigated that immunologically active monocytes/macrophages across the atrial endocardium were more than sinus rhythm atria, and the migrated macrophages expressed TGF-β or IL-6 in atria was more frequent in AF. These findings together strongly have shown the hypothesis that autoimmune and inflammation-regulation mechanism plays an important role in the pathogenesis of the AF in humans.

IL-17 was described as a pro-inflammatory cytokine. In a rat model with sterile pericarditis (SP), the treatment with anti-IL-17A monoclonal antibody significantly reduced the incidence of AF, the number and duration of AF episodes, and the probability of AF induction. Decreased levels of atrial IL-17A could prolong refraction and markedly suppress the development of AF by alleviating inflammation and fibrosis of atria (Fu et al., 2015). Meanwhile the decreased expressions of AF-related pro-inflammatory cytokines and downregulation of fibrosis-related mRNA expression have also been investigated in SP rat with the neutralization of IL-17A (Fu et al., 2015). IL-27 is known to have anti-inflammatory property in the progression of cardiovascular diseases by inhibiting the development of Th17 cells, by which IL-17 is secreted (Owaki et al., 2005; Peters et al., 2015). Studies in animal model indicated that the lack of IL-27 signaling can lead to an increase of Th17, which secretes IL-17. Zhu et al. (2015) found that IL-27 could alleviate coxsackievirus B3-induced myocarditis through modulating Th17 cell responses in mouse model, which further confirmed that IL-27 has anti-inflammatory property. These observations collectively suggest that the IL-27-IL-17 axis and the modulated autoimmune responses appear to play an important role in some inflammatory heart diseases, including AF.

Functional polymorphisms of many genes are involved in susceptibility of AF. Yamase et al. (2016) have identified three polymorphisms of the proline/serine-rich coiled-coil 1 gene, the zinc finger, and C3HC-type containing 1 gene, which were significantly associated with AF in Japanese individuals. Further studies have shown the polymorphisms of several cytokines such as IL-6 and IL-10, which were associated with the risk of AF (Geng et al., 2014; Zheng et al., 2014). All these findings indicated that the polymorphisms of IL-27 gene may have an effect on the susceptibility to AF.

The present study identified the genetic contribution of two polymorphisms in IL-27, including rs153109 (5′untranslated region) and rs17855750 (Exon 2/missense), between AF patients and controls. Our results indicated that there is a true association between allele G of rs153109 and AF susceptibility. Meanwhile, an increasing trend of GG genotype at SNP rs153109 in AF patients has also been investigated. As the study was the relatively small sample size, further studies recruiting a larger number of samples are warranted to confirm the role of GG genotype of rs153109 as a possible susceptibility factor for AF in Chinese population. Thus far, we do not know whether the SNPs of IL-27 gene affect the amount of IL-27 expression, thereby affecting the downstream signals of IL-27. Interestingly, the polymorphism at rs17855750 locus results in a missense mutation in IL-27. Allele T at this locus is corresponding to amino acid Ser, while G allele is Ala. Different amino acid sequences may cause protein structural and/or functional changes. However, the distributions of genotypes and alleles of rs17855750 showed no difference in cases and controls.

Conclusion

To our knowledge, this is the first study to show the association of IL-27 gene polymorphisms with susceptibility to AF in Chinese Han population. It is becoming increasingly clear that IL-27 is attributable to the etiology of AF. However, because genetic polymorphisms vary greatly among ethnic populations, additional studies in a larger number of AF patients and in different populations could help to exclude a population-oriented association.

Limitations of the Study

One of the limitations is that only two SNPs of IL-27 have been assessed in our study. Further studies are needed to examine more loci to verify the association between IL-27 and AF. In addition, more AF patients also need to be used to clarify the association between these SNPs and AF recurrence risk after catheter ablation. Finally, another issue remains to be addressed in the future whether these genetic polymorphisms influence IL-27 gene expression.

Footnotes

Acknowledgments

This work was supported by the Applied Basic Research Programs of Science and Technology Commission Foundation of Sichuan Province (No. 2015JY0186), and Scientific Research Foundation for the Returned Overseas Chinese Scholars of Sichuan Province (No. 2016064).

Author Disclosure Statement

No competing financial interests exist.

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