CHD, which is defined as the structural abnormality of the heart or blood vessels near the heart, present either at the time of birth or detected later on, is a common cause of peri-natal morbidity and mortality.Reference Mitchell, Korones and Berendes 1 , Reference Pierpont, Basson and Benson 2 Most of the known causes of CHD are sporadic genetic changes, but genes regulating the complex developmental sequence have only been partly elucidated.Reference Srivastava 3 In addition, known ante-natal environmental factors include maternal infections – for example, Rubella – drugs such as alcohol, hydantoin, lithium, and thalidomide, and maternal illness – for example, diabetes mellitus, phenylketonuria, and systemic lupus erythematosus.Reference Chinawa, Obu, Eke and Eze 4
Atrial septal defect is the third most common type of CHD, with an estimated incidence of 56/100,000 live births.Reference Hoffman and Kaplan 5 Included in this group of malformations are several types of atrial communications that allow shunting of blood between the systemic and the pulmonary circulations.Reference Geva, Martins and Wald 6 Most atrial septal defects are sporadic with no identifiable cause. Abnormalities in genes essential for cardiac septation have been associated with atrial septal defect.Reference Maitra, Schluterman and Nichols 7
Ventricular septal defect is a common CHD, which accounts for up to 40% of cardiac anomalies.Reference Hoffman 8 In recent years, multiple genes, mainly genes encoding transcriptional factors, such as TBX5, NKX2.5, and GATA4, have been found to be important for cardiac development and are candidate genes for ventricular septal defect.Reference Garg, Kathiriya and Barnes 9 , Reference Gong, Gottlieb and Collins 10 It seems that genetic factors may play an important role in the pathogenesis of ventricular septal defect.
Cardiac development is a complex biological process requiring the integration of cell commitment, morphogenesis, and excitation–contraction coupling.Reference Schott, Benson and Basson 11 In addition, there are a large number of cytokines involved in embryogenesis.Reference Ostojic, Dubanchet, Chaouat, Abdelkarim, Truyens and Capron 12 Previous research has suggested that immune mechanisms may play a critical role in the development of human congenital defects.Reference Rodriguez-Burgos and Juarez 13
IL-27 is a novel heterodimeric cytokine that consists of IL-27p28 and Epstein–Barr virus-induced gene 3 (EBI3).Reference Murugaiyan and Saha 14 – Reference Devergne, Hummel and Koeppen 16 IL-27, with its gene located on chromosome 16 (16p11), is a new IL-12 family member. It is an early product of activated antigen-presenting cells and triggers Th1 polarisation and IFN-γ production in synergy with IL-12. IL-27 mediates its biological effects through a signal-transducing receptor constituted by WSX-1, also known as TCCR, a class I cytokine receptor, and glycoprotein 130 (gp130), an IL-6-related receptor subunit.Reference Pflanz, Hibbert and Mattson 17 , Reference Zhang, Zhou and Wu 18 The success of pregnancy is a complex process with a large number of immune cytokines involved in the process.Reference Laird, Tuckerman and Li 19 Immunohistochemistry studies showed that IL-27 was expressed at the interface and is involved in maternal tolerance to the foetus and successful pregnancy.Reference Coulomb-L’Hermine, Larousserie, Pflanz, Bardel, Kastelein and Devergne 20 In addition, polymorphisms located on genes regulating the immune response may result in increased susceptibility and/or poorer prognosis in certain individual diseases, especially immune-related diseases.Reference Shi, Zhou, Zhang and Zhang 21 , Reference Tao, Wang and Li 22 As IL-27 plays an important role during embryogenesis, we assumed that polymorphisms in IL27 may be associated with the risk for CHD.
The rs153109 and rs17855750 polymorphisms of IL-27p28 were recently identified. Previous studies have demonstrated their association with chronic obstructive pulmonary disease and allergic rhinitis;Reference Shen, Yuan and Hu 23 , Reference Huang, Liu, Wang, Liu, Yin and Yang 24 however, to date, no studies have examined the role of polymorphisms within the IL27 gene in the development of CHD. In the present study, we assumed that polymorphisms in IL27 may affect the risk for CHD. We have identified two possible variation sites of IL27, including one exon (rs17855750) and one promoter (rs153109) sequence. To determine whether these polymorphisms are associated with susceptibility to CHD, we have analysed their frequencies in genomic DNAs isolated from CHD patients and healthy controls.
Material and methods
Patients
The present study was carried out with the approval of the ethics committee of the West China First University Hospital of Sichuan University, and all the patients gave their written informed consent to participate. A hospital-based case–control study was conducted, including 247 unrelated atrial septal defect patients ranging in age from 1 to 65 years (mean±SD, 29.62±16.34; male/female, 74/173) and 150 unrelated ventricular septal defect patients ranging in age from 1 to 53 years (mean±SD, 16.13±12.28; male/female, 69/81) between June 2008 and October 2012 at the First University Hospital of Sichuan University. The diagnoses of atrial septal defect and ventricular septal defect were based on the patient’s history, physical examination, electrocardiogram, and echocardiogram studies. Clinical data were collected from the hospital record section. A group of control patients, including 368 healthy controls ranging in age from 12 to 48 years (mean±SD, 26.69±6.33; male/female, 135/233) was selected randomly from a routine health survey in the same hospital. Patients with any personal or family history of heart disease or other serious disease were intentionally excluded.
Genotyping
Genomic DNA of each individual was extracted from 200 µl of ethylenediaminetetraacetic acid-anti-coagulated peripheral blood samples using a DNA isolation kit from Bioteke (Peking, China). The procedure was performed according to the instruction manual. The genotypes of the two single nucleotide polymorphisms – rs153109 and rs17855750 – selected were analysed using a polymerase chain reaction-restriction fragment length polymorphism assay. The primers used for amplification of the rs153109 polymorphism were F: 5'-CTGATCCTGACCTCACTCAACGC-3' and R: 5'-CTGACTGGGACTGGGACTCAGC-3', and primers used for amplification of the rs17855750 polymorphism were F: 5'-ATCTCGCCAGGAAGCTGCGC-3' and R: 5'-CTGTTAGTGGGGGCCAGAAGGGA-3'. The polymerase chain reaction reactions were performed in a total volume of 25 µl, including 2.5 µl 10× polymerase chain reaction buffer, 1.5 mmol/L MgCl2, 0.15 mmol/L dNTPs, 0.5 µmol/L each primer, 100 ng of genomic DNA, and 1U of Taq DNA polymerase. The polymerase chain reaction conditions for rs153109 were as follows: 94°C for 4 minutes, followed by 32 cycles of 30 seconds at 94°C, 30 seconds at 60°C, and 30 seconds at 72°C, with a final elongation at 72°C for 10 minutes; and the polymerase chain reaction conditions for rs17855750 were as follows: 94°C for 4 minute, followed by 36 cycles of 30 seconds at 94°C, 30 seconds at 66°C, and 30 seconds at 72°C, with a final elongation at 72°C for 10 minutes. Polymerase chain reaction products of these two polymorphisms were digested overnight with a specific restriction enzyme, Bstu I, and the digested polymerase chain reaction products were separated using 6% polyacrylamide gel and stained with 1.0 mg/ml argent nitrate: for rs153109, allele G was cuttable, yielding two fragments of 100 and 19 bp, allele A was uncuttable and the fragment is still 119 bp. For rs17855750, allele G was cuttable, yielding two fragments of 101 and 19 bp, allele T was uncuttable and the fragment is still 120 bp. Approximately 20% of the samples were randomly selected to carry out the repeated assays and the results were 100% concordant. The genotypes were confirmed by DNA sequencing analysis.
Statistical analysis
All data analyses were carried out using SPSS 13.0 statistical software (SPSS Inc., Chicago, Illinios, United States of America). Allele and genotype frequency of the IL27 gene rs153109 and rs17855750 were obtained by directed counting, and the Hardy–Weinberg equilibrium was evaluated by the χ2 test. Genotypic association tests in a case–control pattern assuming co-dominant, dominant, recessive, over-dominant, or log-additive genetic models were performed using SNPstats.Reference Sole, Guino, Valls, Iniesta and Moreno 25 Odds ratio and respective 95% confidence intervals were reported to evaluate the effects of any difference between alleles and genotypes. A p<0.05 was regarded as statistically significant.
Results
Both rs153109 and rs17855750 polymorphisms were successfully genotyped in 247 atrial septal defect patients and in 368 healthy control patients. Genotype distribution of these two polymorphisms in our cases and controls were consistent with the Hardy–Weinberg equilibrium. Allele frequencies of these two polymorphisms for 247 atrial septal defect patients and for 368 control patients are shown in Table 1. Significantly increased atrial septal defect risk was observed to be associated with the allele G of rs153109 locus (p=0.001, OR=1.490, 95% CI=1.178–1.887); however, the allele frequency of rs17855750 polymorphism was not significantly different between atrial septal defect patients and controls (p=0.139, OR=0.747, 95% CI=0.510–1.094)
Table 1 Data of selected polymorphisms in the IL27 gene among patients with atrial septal defect and controls.
CI=confidence interval; OR=odds ratio
No corresponds to the number of individuals
Boldfaced values indicate a significant difference at the 5% level
As shown in Table 2, significantly increased atrial septal defect risk was found to be associated with the AG (p<0.01, OR=1.89, 95% CI=1.32–2.70) and GG genotypes (p<0.01, OR=1.89, 95% CI=1.15–3.03) of the rs153109 polymorphism in the co-dominant model, compared with the AA genotype. Compared with the homozygote AA/GG genotypes carriers, the AG heterozygote carriers have a 1.59-fold atrial septal defect risk in the over-dominant model (p<0.01, OR=1.59, 95% CI=1.15–2.17). Moreover, in the dominant model, significantly increased atrial septal defect susceptibility was also observed to be associated with G allele carriers (p<0.01, OR=1.89, 95% CI=1.35–2.63); however, for genotypic association analysis, no statistically significant difference was detected between atrial septal defect patients and control patients for the rs17855750 polymorphism.
Table 2 Genotype frequencies of selected polymorphisms in the IL27 gene among patients with atrial septal defect and controls and their association with atrial septal defect risk.
CI=confidence interval; OR=odds ratio
No corresponds to the number of individuals
Boldfaced values indicate a significant difference at the 5% level
* Adjusted by age
To further investigate whether these two polymorphisms of the IL27 gene are associated with other types of CHD, we also detected these two polymorphisms of the IL27 gene on ventricular septal defect patients. Allele frequencies of these two polymorphisms for 150 ventricular septal defect patients and 368 controls are shown in Table 3. Significantly increased ventricular septal defect risk was observed to be associated with the G allele of rs153109 locus (p=0.004, OR=1.502, 95% CI=1.139–1.976), but allele frequency of the rs17855750 polymorphism was not significantly different between ventricular septal defect patients and controls (p=0.809, OR=0.947, 95% CI=0.590–1.518). As shown in Table 4, significantly increased ventricular septal defect risk was found to be associated with the AG genotype of the rs153109 polymorphism in the co-dominant model, compared with AA and GG genotypes (p<0.01, OR=2.86, 95% CI=1.85–4.35). Compared with the homozygote AA/GG genotypes carriers, the AG heterozygote carriers have a 2.56-fold ventricular septal defect risk in the over-dominant model (p<0.01, OR=2.56, 95% CI=1.72–3.85). Moreover, in the dominant model, significantly increased ventricular septal defect susceptibility was also observed to be associated with G allele carriers (p<0.01, OR=2.50, 95% CI=1.67–3.85); however, for genotypic association analysis, no statistically significant difference was detected between atrial septal defect patients and controls for the rs17855750 polymorphism.
Table 3 Data of selected polymorphisms in the IL27 gene among patients with ventricular septal defect and controls.
CI=confidence interval; OR=odds ratio
No corresponds to the number of individuals
Boldfaced values indicate a significant difference at the 5% level
Table 4 Genotype frequencies of selected polymorphisms in the IL27 gene among patients with ventricular septal defect and controls and their association with ventricular septal defect risk.
CI=confidence interval; OR=odds ratio
No corresponds to the number of individuals
Boldfaced values indicate a significant difference at the 5% level
* Adjusted by age
Discussion
To our knowledge, this is the first study to investigate the association between the polymorphisms in the IL27 gene and CHD risk. Our results demonstrated that the allele G of the rs153109 polymorphism may increase atrial septal defect and ventricular septal defect susceptibility. We have calculated the statistical power with “Power and Sample Size Calculation” software (version 3.0.43), and our study had >80% power to detect the association between this polymorphism of IL27 and both atrial septal defect and ventricular septal defect susceptibility.The rs17855750 polymorphism, however, has no statistically significant differences between CHD patients, including atrial septal defect and ventricular septal defect patients, and healthy control patients.
CHD, a heart problem caused by the improper development of the heart during foetal development, is always presented at birth.Reference Chinawa, Obu, Eke and Eze 4 It has been known as a major cause for infant mortality, but the aetiology of the vast majority of CHD is not fully addressed. Cardiac development is a complex biological process requiring the integration of cell commitment, morphogenesis, and excitation–contraction coupling.Reference Schott, Benson and Basson 11 Both genetic and environmental factors are suspected to be involved.
As we know, a large number of immune cytokines play an important role in either the progress of pregnancy or implantation.Reference Mas, Petitbarat, Dubanchet, Fay, Ledee and Chaouat 26 Increased risk for atrioventricular septal defects has been observed to be associated with maternal urinary tract infections in the first trimester.Reference Cleves, Malik, Yang, Carter and Hobbs 27 Moreover, dysregulation of the maternal immune response related to an elevation of cytokines important in inflammatory conditions is suggested to play a role in cardiac malformations during foetal development.Reference Blossom, Rau, Best, Bornemeier and Hobbs 28 In addition, cytokines have been confirmed to be involved in various cardiovascular diseases.Reference Mann and Young 29 – Reference Werdan 31 Previous studies have suggested the potential association between the aberration in cytokine production and CHD risk.Reference Blossom, Rau, Best, Bornemeier and Hobbs 28 , Reference Buchhorn, Wessel, Hulpke-Wette, Bursch, Werdan and Loppnow 32
IL-27, a pro-inflammatory cytokine, composed of Epstein–Barr virus-induced gene 3 protein (EBI3) and p28, plays an important role in response to infection, and is expressed at a very high level at the human foetal–maternal interface.Reference Devergne, Hummel and Koeppen 16 , Reference Coulomb-L’Hermine, Larousserie, Pflanz, Bardel, Kastelein and Devergne 20 , Reference Devergne, Birkenbach and Kieff 33 – Reference Gee, Guzzo, Che Mat, Ma and Kumar 36 Existing research has found that IL-27 acts directly on endothelial cells and inhibits angiogenesis by inducing the production of anti-angiogenic chemokines IP-10 and MIG.Reference Shimizu, Shimamura and Owaki 37 Moreover, IL-27 produced by extravillous trophoblasts may prevent excess pro-angiogenic activity through a negative-feedback mechanism.Reference Coulomb-L’Hermine, Larousserie, Pflanz, Bardel, Kastelein and Devergne 20 Taken together, there may be an association between IL-27 and angiogenesis during embryogenesis, and thus IL-27 may be involved in the pathogenesis of CHD.
Existing researches have suggested an important role genetic factors may play in the pathogenesis of CHD.Reference Srivastava 3 , Reference Chinawa, Obu, Eke and Eze 4 Polymorphisms in genes potentially influence susceptibility to CHD. Identification of human mutations that cause CHD offers a complementary approach to gene ablation studies and particularly fosters definition of gene defects that perturb later stages of cardiac development.Reference Schott, Benson and Basson 11 As IL-27 may play an important role in the pathogenesis of CHD, genetic variation in the IL27 gene may be associated with risk for this disease. In the present study, our data demonstrated an association between rs153109 polymorphism and susceptibility to CHD, including atrial septal defect and ventricular septal defect. These findings suggested that the rs153109 polymorphism may be a contributor to the pathogenesis of CHD. This polymorphism might become a useful prognostic biomarker for CHD patients. Nonetheless, the precise role the IL27 gene may have in the development of CHD remained unclear, further studies are necessary to confirm our findings and its possible mechanisms.
Although we detected the association between rs153109 polymorphism in IL27 and CHD, it is worth mentioning that there are some limitations to our study. First, we did not detect the protein level of IL-27 in the peripheral blood and did not perform a functional analysis study, thus we could not draw a certain conclusion about the influence of these polymorphisms on the cytokine levels. Second, the number of patients in our study were limited. Further large-scale studies are necessary to confirm our findings.
In summary, the present study demonstrated that the allele G and GG genotype of the rs153109 polymorphism in the IL27 gene contribute to increased CHD susceptibility, including atrial septal defect and ventricular septal defect, in Chinese Han population. Nevertheless, further studies are necessary to investigate the real association between these two polymorphisms and the risk for CHD, especially in ethnically disparate population.
Financial Support
This work was supported by the fund of Chongqing Population and Family Planning Science and Technology Research Institute (No.1304, No.1202); 2013cstc-jbky-01704; 2012cstc-jbky-01702; CSTC,2009CA5001.
Conflicts of Interest
None.
