Introduction: The chronic inflammatory state in atherosclerosis is an important risk factor for cardiovascular (CV) outcomes. The influence of the genetic polymorphism of interferon (IFN)-gamma on such outcomes needs to be evaluated.

Objective: The aim of this study is to investigate the association between genetic polymorphism of IFN-gamma and the risk of CV event and/or death.

Patients and Methods: A prospective study was conducted including 208 hypertensive patients, where the polymorphism of IFN-gamma (+874 A → T) was analyzed. We evaluated as outcomes the occurrence of a CV event with (combined endpoint) or without death and the association between IFN-gamma polymorphism and the outcomes was analyzed.

Results: The patients had a mean age of 59.7 ± 9.3 years, with 57.2% being white, 37% having type 2 diabetes mellitus (DM) and 59.1% having metabolic syndrome (MS). Regarding IFN-gamma polymorphism, 17.7% had a TT genotype, 37.5% a TA genotype and 44.8% an AA genotype. After the follow-up, there were 7 deaths and 8 CV events. The frequency of the TT genotype was higher in patients who had a CV event (60 versus 15% in those who did not) and combined endpoint (55.6% versus 15%). Even after adjusting for age, gender, DM, total cholesterol, uric acid and MS, the presence of the TT genotype was an independent predictor for the occurrence of a CV event (RR: 10.12, CI: 1.16 – 87.79, p < 0.05) and combined outcome (RR: 10.74, CI: 1.909 – 60.47, p < 0.05).

Conclusion: In individuals with hypertension, the presence of the TT genotype of the IFN-gamma polymorphism resulted in a higher incidence of CV event and death.

•    Hypertension
•    Polymorphism
•    Interferon-gamma
•    Cardiovascular disease
•    Inflammation

Coutinho SM, Triches CB, Redublo Quinto BM, de Oliveira Rodrigues CJ, Batista MC (2016) Influence of Genetic Polymorphism of IFNGamma on the Occurrence of Cardiovascular Events in Patients with Hypertension. Ann Clin Exp Hypertension 4(2): 1037.

BMI – Body Mass Index; CAD - Coronary Artery Disease; CKD – Chronic Kidney Disease; CKDEPI - Chronic Kidney Disease Epidemiology Collaboration; CRP – C Reactive Protein; CV – Cardiovascular; CVD – Cardiovascular Disease; DBP – Diastolic Blood Pressure; DM – Diabetes Mellitus; eGFR – estimated Glomerular Filtration Rate ESRD – End Stage Renal Disease; FRS – Framingham Risk Score; HDL – High Density Lipoprotein; IDF - International Diabetes Federation; IFN – Interferon; IL – Interleukin; LDL – Low Density Lipoprotein; MHC – Main Histocompatibility Complex; MS – Metabolic Syndrome; NFKB – Nuclear Factor kappa B; PAD – Peripheral Arterial Disease; ROS – Reactive Oxygen Species; SAH – Systemic Arterial Hypertension; SBP – Systolic Blood Pressure; SNP – Single Nucleotide Polymorphism; TGD – Triglycerides; TGF – Transforming Growth Factor; TNF – Tumor Necrosis Factor

Recognized as an important risk factor for cardiovascular disease (CVD), systemic arterial hypertension (SAH) interacts with various risk factors in the development of atherosclerosis in the coronary and cerebral arteries and the association between hypertension and inflammation results in considerable increase in coronary artery disease (CAD) risk [1]. Accordingly, chronic inflammation, involved in the genesis and perpetuation of atherosclerotic disease, is associated with increased cardiovascular risk [2-4], and serum levels of several inflammatory cytokines (IL-6, TNF, IL-10, TGF, IFN-gamma) have been shown to be elevated in individuals with established atherosclerotic cardiovascular disease [5-6]. The serum levels of C-reactive protein (CRP), a systemic marker of inflammation, predicts risk of coronary events adding predictive power to the isolated measurement of lipid profile, blood glucose and other risk factors [7].

Interferon (IFN)-gamma is a T cell-derived cytokine and serves as a marker for Th1 helper lymphocyte activation, which promotes and amplifies the inflammatory response, thereby playing an important role in the coordinate regulation of the immune response. IFN also has direct effects on inflammation by increasing the expression of the major Histocompatibility complex (MHC) class II and stimulates macrophages to produce inflammatory mediators of tissue damage, such as TNF alpha, proteases, reactive oxygen species (ROS) and nitric oxide [8]

In the general population, IFN-gamma is highly expressed in atherosclerotic lesions and has emerged as a significant factor in the development and progression of CVD. Studies have shown that IFN is the major trigger for the production and release of ROS also in the endothelium and that a T cell subtype involved in atherosclerosis produces large amounts of IFN-gamma [9]. Chronic ROS production leads to depletion of antioxidants such as vitamin C, vitamin E and glutathione, with consequent development of oxidative stress. This condition is the main factor responsible for atherogenesis and progression of CVD [9].

It is known that the genetic control of cytokine production is affected by various known polymorphisms, which may result in higher or lower levels of these inflammatory mediators in plasma. A single nucleotide polymorphism (SNP) at position +874 of the IFN-gamma gene (+874 T → A) may influence the secretion of IFN-gamma [10]. Studies of the biological role of this polymorphism have suggested that allele a carriers are low producers of IFN-gamma [11]. The presence of the T allele in this functional variant increases NFKB binding efficacy, leading to a high expression of IFN-gamma in vitro [10]. The polymorphism at position +874

(A → T) has been associated with increased expression of IFN-gamma and greater susceptibility to chronic kidney disease (CKD) for various reasons [12]. Thus, this study aimed to elucidate the potential association between the IFN-gamma polymorphism +874 T → A and the occurrence of the following outcomes:

1. Primary: cardiovascular (CV) events and death

2. Secondary: progression of CKD and albuminuria and incidence of DM and metabolic syndrome (MS).

A total of 664 consecutive patients seen at the outpatient clinic of the Integrated Center for Hypertension and Cardiovascular Metabolism of the Oswald Ramos Foundation-UNIFESP were considered, and only patients diagnosed with SAH were included for cardiovascular risk stratification and genetic study.

We collected the following data from medical charts: age, sex, race, weight, height, body mass index (BMI), systolic (SBP) and diastolic (DBP) blood pressure, diagnosis of DM, obesity and CVD, family history, smoking history, fasting blood glucose, total cholesterol and fractions, triglycerides, waist circumference, diagnosis of metabolic syndrome (MS) by the criteria of the International Diabetes Federation (IDF) [13], creatinine, uric acid, CRP, albuminuria in a single sample, glomerular filtration rate estimated by the formula of the CKD-EPI (eGFR CKD-EPI) (14), and the Framingham Risk Score [15]. The data of IFNgamma polymorphism (+874 T→ A) were also recorded. BMI was calculated by the formula: weight (kg)/ (height x height) (m).

CVD was defined as the presence of CAD (evidence of silent myocardial infarction or myocardial ischemia, history of unstable or stable angina, and coronary angioplasty or coronary surgery), or equivalent risk of CAD, stroke, peripheral arterial disease (PAD), abdominal aortic aneurysm, carotid artery disease or renal artery disease, according to the American Heart Association [16].

For analysis of lipid profile, blood glucose, CRP and uric acid, blood samples were drawn after a 12-hour period of fasting. Plasma concentrations of glucose, total cholesterol and triglycerides were determined by automated enzymatic methods. The measurement of HDL-cholesterol and LDLcholesterol fractions was performed using commercial kits according to manufacturer’s specifications. Plasma CRP and uric acid were determined using a commercial kit according to the manufacturer’s specifications. Serum creatinine levels were measured using the alkaline picrate method traceable to isotope dilution mass spectrometry. Albuminuria was measured with immune turbidimetry and normal value was defined as < 26mg/g.

Analysis of cytokine gene polymorphisms

Genomic DNA was isolated from peripheral blood using standard techniques. The SNP 874T/A was detected by PCR (polymerase chain reaction) using the cytokine genotyping kit (One Lambda, Canoga Park, CA, USA) according to the manufacturer’s instructions. The genotyping kit allowed the detection of the +874 IFN -gamma gene polymorphism (T versus A). Besides the specific primer for IFN-gamma, internal oligonucleotide sequences were added as a positive control for each reaction. The primer of internal control amplifies a region of the beta-globin gene of 750 bp. This internal control was present in each well.

The PCR product was analyzed by 2% agarose gel electrophoresis, and the specific amplification bands were visualized by UV light and photographed for later analysis of the polymorphism. Quality checks to ensure the reliability of genotyping were carried out by an independent evaluation of the results by two or three investigators. Discrepancies were all resolved through consensus or redoing genotyping.

After a period of 71.2 ± 15 months (5.8 ± 1.2 years) the results of the same laboratory tests and physical examination were collected again for the assembly of a comparative database. Those patients who had incomplete data or who were lost to follow-up were excluded. The remaining 208 patients were included in the prospective study. The incidence of new CV events or death was identified through medical records of CAD, stroke, PAD or other atherosclerotic vascular events. We also evaluated progression of CKD through decrease in eGFR, increase in albuminuria, increase in cardiovascular risk through the Framingham Risk Score, and control of blood pressure, DM and dyslipidemia, as well as the incidence of obesity and MS. IFN-gamma polymorphism was associated with the new data and outcomes obtained to establish risk association.

Statistical analysis

Data were expressed as mean and standard deviation for variables with normal distribution and median with minimum and maximum values for non-parametric variables. The Student t-test (parametric variables) and the Mann-Whitney test (nonparametric variables) were used to evaluate differences in the various variables. The chi-square test was used to compare proportions between groups with and without outcomes analyzed. A binary logistic regression model was used to confirm the associations found by adjusting them for confounding factors. We used p<0.05 for rejection of the null hypothesis in all tests. SPSS 20.0 software was used for statistical analysis.

The population was predominantly female (70.2%), while the majority (57.2%) was Caucasian, and all patients were hypertensive. The mean age of patients was 59.7 ± 9.3 years; 37% had type 2 DM, while 59.1% had MS, and the mean BMI was 30.17 ± 6.0 cm (Table 1).

Figure 1 Frequency of IFN –gamma Genotypes (%)

With regard to IFN-gamma polymorphism, 17.7% of patients had the TT genotype, 37.5% the TA genotype and 44.8% the AA genotype (Figure 1). The observed genotype distributions were in Hardy-Weinberg equilibrium. For the presence of each allele alone, 82.3% were carriers of the A allele and 55.1% of the T allele (Figure 2). There was no significant difference in the distribution of studied IFN-gamma genotypes with respect to ethnic group (Table 2).

Figure 2 Frequency of T and A Alleles in the population (%).

At the end of follow-up (after 5.8 ± 1.2 years) we observed a better control of DBP (82.0 ± 10.2 mmHg vs. 84.6 ± 10.7 mmHg), higher fasting blood glucose levels [98 (30-368) mg/dl versus 94.5 (67-381) mg/dl] and increased waist circumference (102.3 ± 13.5 versus 97.6 ± cm 13.2 cm) compared to baseline, all of them with statistical significance (p < 0,05) (Table 1). Patients also had lower total cholesterol levels (180.5 ± 38.9 mg/dl versus 198.8 ± 41.2 mg/dl) and HDL (47.1 ± 12.1 mg/dl versus 54.5 ± 15.5 mg/ dl), both with p < 0,05 (Table 1). The incidence of DM in the study population was 15%, while incidence of obesity was 3% and MS was 19%, statistically significant. The frequency of patients with eGFR <60 ml/min increased to 17.4% and there was an increase in albuminuria (9.2 (0 to 2893.0) mg/g versus 4.4 (0 to 525.0) mg/g) at the end of follow-up, both with statistical significance (Table 1). The Framingham Risk Score increased to 11.3 ± 8.8% (versus 7.9 ± 7% at the beginning), consistent with the aging of the population (Table 1). The number of antihypertensive agents used by individuals increased during the follow up (2.0 ± 0.4 to 2.3 ± 1.3 – p < 0.05) (Table 1).

We divided population according to IFN polymorphism (A allele carriers versus TT genotype) and found no differences in groups profiles with respect to baseline characteristics (Table 3). Only a tendency toward higher prevalence of obesity and albuminuria > 26mg/g in the TT group was observed (Table 3). After follow-up, however, this trend was not evident (data not shown).

Seven patients died (death from all causes), and new CV events occurred in 8 patients (4 of whom suffered a myocardial infarct and 4 of whom were diagnosed with stroke).

No significant associations were found between IFN-gamma polymorphism and decrease in eGFR, increase in albuminuria, and incidence of DM and MS (Table 3).

The frequency of the TT genotype was higher in patients with CV event (60 versus 15%, p < 0,05 – Table (4) and was also higher in those with combined outcome - death or CV event (55.6 versus 15%, p < 0,05 – Table (5).

After logistic regression analysis, adjusted for age, gender, DM, total cholesterol, uric acid and MS, the presence of the TT genotype was an independent predictor for the occurrence of CV event (RR: 10.12, CI: 1.16– 87.79, p < 0.05) and combined outcome - death or CV event (RR: 10.74, CI: 1.909 – 60.47, p < 0.05 – Table (6).

Table 1: Characteristics of the population before and after follow-up. Duration in months (mean ± SD): 71.2 ± 15 months

Table 2: Distribution of IFN-gamma genotypes in the population according to race (p= NS).

Table 3: Characteristics of the population in relation to IFN-gamma polymorphism.

The influence of IFN-gamma in cardiovascular risk has been the subject of several studies [17-25]. The role of chronic inflammation such as atherosclerotic disease predictor is what justified the present study. Some known cardiovascular risk factors can be controlled, either through changes in lifestyle or drug therapy. However, given the failure to modify the genetic risk factors, the study of IFN polymorphism appears to be extremely relevant, even to contribute to the justification of family history of early CAD as one of the classical cardiovascular risk factors.

Inflammatory response is modulated by the balance between pro- and anti-inflammatory mediators. IFN-gamma is a proinflammatory cytokine that plays a crucial role in host defense by directing the immune response to pro-inflammatory mediators including TNF-alpha and IL-6 [26]. Due to these functional characteristics, the bioactivity of IFN-gamma has been identified as a key mediator in several models of inflammatory diseases [21- 26]. Individuals with certain genotypes are commonly referred to as low (AA), intermediate (AT) or high (TT) IFN-gamma producers, depending on their production rate in vitro. However, there is considerable inter-individual variation in the degree of systemic inflammatory activation, because the production of cytokines is in part genetically determined.

In a cohort of hypertensive patients, we demonstrated that patients with the TT genotype in the IFN-gamma polymorphism had a higher risk of death and CV event at the end of follow-up of 5.8 years. Similarly, Manginas and colleagues [20] showed a predominance of T allele in patients with unstable angina or myocardial infarction compared with patients with stable angina, also suggesting that T allele patients are more prone to serious CV events.

The works of Biolo [21] and Tripathi [12] showed that the genotypes (IFN-gamma +874) of intermediate (TA) and high (TT) producers were significantly associated with progression of CKD, while the low producer (AA) was associated with a protective effect.

In our population, however, there were no significant associations of IFN-gamma polymorphism (+874 T → A) with the decrease in eGFR or the increase in albuminuria.

Moreover, Adamopoulos et al. [22] found that in 80 patients with idiopathic dilated cardiomyopathy, those with the AA genotype (compared to T allele carriers) showed more deaths from cardiac causes or need for heart transplantation. Torres et al., [27] also demonstrated a higher prevalence of the AA genotype and carriers of the A allele in patients with Changes disease compared to healthy patients, suggesting a greater susceptibility to infection with Trypanosoma cruzi, but did not establishing a relationship with the progression of Chagas cardiomyopathy.

Notably, to date, there is a great divergence between studies with respect to which polymorphism shows higher cardiovascular risk. In a Spanish study, Garcia-Bermudez and coworkers [28] genotyped (IFN-gamma + 874 T → A) 1635 patients with rheumatoid arthritis and stratified them according to the presence or absence of CV events. No genotype or allele was associated with the presence of CV events in this population.

The present study had some limitations. Only 208 individuals were included in the prospective analysis, due to loss of information or follow-up, which resulted in a small number of outcomes (7 deaths and 8 CV events) and decreased the statistical power. Deaths were considered from all causes (not just related to CV events), because there was no record of the death cause in the medical records and because there was no access to death certificates. The method used for genetic analysis in this study (SNP) has limitation in understanding the interface genotype: phenotype, which is part of the genetic predisposition; and this method does not allow the simultaneous analysis of several genes, such as in whole genome analysis, thereby hindering the search for the genetic signature for polygenic diseases such as CVD and CKD.

Our study established a clear association of the TT genotype with increased cardiovascular risk in comparison with other genotypes, so it has to be regarded as a promising risk factor to be investigated.

New prospective studies with larger population sample are necessary to determine if the genetic polymorphism of IFN-gamma may contribute to the elucidation of the complex interactions involved in the pathogenesis of CVD and to the identification of targets for therapeutic interventions.

Table 4: Baseline clinical and laboratory characteristics of the population with cardiovascular event (+) and without cardiovascular event (-).

Table 5: Baseline clinical and laboratory characteristics of the population with death or cardiovascular. Cardiovascular event (+) and without death or cardiovascular event (-).

 Table 6: Association of genotype TT and risk of cardiovascular event or death.

In individuals with hypertension, the presence of the TT genotype of the IFN-gamma polymorphism resulted in a higher incidence of CV event and death.

Nephrology Division – Universidade Federal de São Paulo / UNIFESP.

1. Gaziano JM, Sesso HD, Breslow JL, Hennekens CH, Buring JE. Relation between systemic hypertension and blood lipids on the risk of myocardial infarction. Am J Cardiol. 1999; 84: 768-773.

2. Ridker PM, Buring JE, Cook NR, Rifai N. C-reactive protein, the metabolic syndrome and risk of incident cardiovascular events: a 8-year follow-up of 14.719 initially health American women. Circulation. 2003; 107:391-397.

3. Sattar N, Gaw A, Scherbakova O, Ford I, O’Reilly DS, Haffner SM, et al . Metabolic syndrome with and without C-reactive protein as a predictor of coronary heart disease and diabetes in West of Scotland Coronary prevention Study. Circulation. 2003; 108: 414-419.

4. Rutter MK, Meigs JB, Sullivan LM, D’Agostino RB, Wilson PW. C-reactive protein, the metabolic syndrome and prediction of cardiovascular events in the Framingham Offspring Study. Circulation. 2004; 110: 380-385.

5. Yudkin JS, Kumari M, Humphries SE, Mohamed-Ali V. Inflammation, obesity, stress and coronary heart disease: is interleukin-6 the link?. Atherosclerosis. 2000; 148: 209-214.

6. Berg KK, Madsen HO, Garred P, Wiseth R, Gunnes S, Videm V, et al. The additive contribution from inflammatory genetic markers on the severity of cardiovascular disease. Scand J Immunol. 2009; 69: 36-42.

7. Maggi E, Marchesi E, Ravetta V, Falaschi F, Finardi G, Bellomo G. Lowdensity lipoprotein oxidation in essential hypertension. J Hypertens. 1993; 11: 1103-1111.

8. Firestein GS. Immunologic mechanisms in the pathogenesis of rheumatoid arthritis. J Clin Rheumatol. 2005; 11: 39–44.

9. Schroecksnadel K, Frick B, Winkler C, Fuchs D. Crucial role of interferon-gamma and stimulated macrophages in cardiovascular disease. Curr Vasc Pharmacol. 2006; 4: 205–213.

10. Pravica V, Perrey C, Stevens A, Lee JH, Hutchinson IV. A single nucleotide polymorphism in the first intron of the human IFN-gamma gene: absolute correlation with a polymorphic CA microsatellite marker of high IFN-gamma production. Hum Immunol. 2000; 61, 863–866.

11. Lopez Maderuelo D, Arnalich F, Serantes R, Gonzalez A, Codoceo R, Madero R, et al. Interferon-gamma and interleukin-10 gene polymorphisms in pulmonary tuberculosis. Am J Respir Crit Care Med. 2003; 167: 970–975.

12. Tripathi G, Borkar M, Akhter A, Sankhwar SN, Sharma RK, Agrawal S, et al. Association of proinflammatory cytokines with end stage renal disease. Cytokine. 2010; 50: 278–283.

13. The IDF consensus worldwide definition of the metabolic syndrome. 2006; International Diabetes Federation.

14. Levey AS, Stevens LA, Schmid CH, Zhang YL, Castro AF 3rd, Feldman HI, et al. A New Equation to Estimate Glomerular Filtration Rate. Ann Intern Med. 2009; 150: 604-612.

15. D Agostino RB , Vasan RS, Pencina MJ, Wolf PA, Cobain M, Massaro JM, et al. General cardiovascular risk profile for use in primary care: the Framingham Heart Study. Circulation. 2008; 117: 743-753.

16. Thom T, Haase N, Rosamond W, Howard VJ, Rumsfeld J, Manolio T, et al. American Heart Association Statistics Committee and Stroke Statistics Subcommittee. Heart disease and stroke statistics—2006 update: a report from the American Heart Association Statistics Committee and Stroke Statistics Subcommittee. Circulation. 2006; 113:e85–e151.

17. Yilmaz G, Arumugam TV, Stokes KY, Granger DN. Role of T lymphocytes and interferon-gamma in ischemic stroke. Circulation. 2006; 113: 2105-2012.

18. Densem CG, Hutchinson IV, Yonan N, Brooks NH. Influence of IFNgamma polymorphism on the development of coronary vasculopathy after cardiac transplantation. Ann Thorac Surg. 2004; 77: 875-880.

19. Lambertsen KL, Gregersen R, Meldgaard M, Clausen BH, Heibol EK, Ladeby R, et al. A role for interferon-gamma in focal cerebral ischemia in mice. J Neuropathol Exp Neurol. 2004; 63: 942-955.

20. Manginas A, Tsiavou A, Chaidaroglou A, Giamouzis G, Degiannis D, Panagiotakos D, Cokkinos DV. Inflammatory cytokine gene variants in coronary artery disease patients in Greece. Coron Artery Dis. 2008; 19: 575-582.

21. Gianni Biolo, Antonio Amoroso, Silvana Savoldi, Alessandra Bosutti, Massimiliano Martone, Doroti Pirulli, et al. Association of interferon-c 1874A polymorphism with reduced long-term inflammatory response in haemodialysis patients. Nephrol Dial Transplant. 2006; 21: 1317– 1322.

22. Adamopoulos S1, Kolokathis F, Gkouziouta A, Georgiadou P, Chaidaroglou A, Karavolias GK, et al. Cytokine gene polymorphisms are associated with markers of disease severity and prognosis in patients with idiopathic dilated cardiomyopathy. Cytokine. 2011 ; 54: 68-73.

23. Raisarnen Sokolowzki A, Glysing Jensen T, Koglin J, Russell ME. Reduced transplant artheriosclerosis in murine cardiac allografts placed in interferon gamma knockout recipients. An J Pathol. 1998; 152: 359-365.

24. Kim HJ, Kang SW, Chung JH, Kim SJ, Choe BK. Polymorphisms of the Interferon gamma gene and coronary artery disease in the Korean population. Mol Biol Rep. 2012; 39: 5425-5432.

25. Babu BM, Reddy BP, Priya VH, Munshi A, Rani HS, Latha GS, et al. Cytokine gene polymorphisms in the susceptibility to acute coronary syndrome. Genet Test Mol Biomarkers. 2012; 16: 359-365.

26. Billiau A. Interferon gamma: biology and role in pathogenesis. Adv Immunol. 1996; 62: 61–130.

27. Torres OA, Calzada JE, Beraún Y, Morillo CA, González A, González CI, et al. Role of the IFNG +874T/A polymorphism in Chagas disease in a Colombian population. Infect Genet Evol. 2010; 10: 682-685.

28. García Bermúdez M, López Mejías R, González Juanatey C, Corrales A, Robledo G, Castañeda S, et al. Analysis of the interferon gamma (rs2430561, +874T/A) functional gene variant in relation to the presence of cardiovascular events in rheumatoid arthritis. PLoS One. 2012; 7: 47166.