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The MHC2TA 1614 C>G gene polymorphism is associated with risk of developing acute coronary syndrome
Molecular Immunology 55 (2013) 424–428
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Molecular Immunology journal homepage: www.elsevier.com/locate/molimm
Short communication
The MHC2TA 1614 C>G gene polymorphism is associated with risk of developing acute coronary syndrome b ˜ Gilberto Vargas-Alarcón a , Marco Antonio Martínez-Ríos b , Marco Antonio Pena-Duque , c c d ˜ , Maite Vallejo , Julián Ramírez-Bello e , Carlos Posadas-Romero , Guillermo Cardoso-Saldana Oscar Pérez-Méndez a , José Manuel Fragoso a,∗ a
Department of Molecular Biology, Instituto Nacional de Cardiología Ignacio Chávez, Mexico City, Mexico Department of Interventional Cardiology, Instituto Nacional de Cardiología Ignacio Chávez, Mexico City, Mexico c Department of Endocrinology, Instituto Nacional de Cardiología Ignacio Chávez, Mexico City, Mexico d Department of Sociomedicine, Instituto Nacional de Cardiología Ignacio Chávez, Mexico City, Mexico e Immunogenomics and Metabolic Diseases Laboratory, Instituto Nacional de Medicina Genómica, Mexico City, Mexico b
a r t i c l e
i n f o
Article history: Received 11 February 2013 Received in revised form 21 February 2013 Accepted 25 February 2013 Available online 16 March 2013 Keywords: Acute coronary syndrome Genetic susceptibility Histocompatibility complex class II trans-activator (MHC2TA) Polymorphism
a b s t r a c t Background: Inflammation plays an essential role in the development and progression of atherosclerotic lesions. The major histocompatibility complex class II trans-activator (MHC2TA) is considered an important molecule in the inflammatory process regulation. The aim of the present study was to evaluate the role of MHC2TA gene polymorphisms as susceptibility markers for acute coronary syndrome (ACS). Methods: Three polymorphisms (−168 A>G, 1614 C>G, and 2536 G>A) of the MHC2TA gene were analyzed by 5 exonuclease TaqMan genotyping assays in a group of 297 patients with ACS and 283 healthy controls. Haplotypes were constructed after linkage disequilibrium analysis. Results: The 1614 C allele and CC genotype were associated with risk of developing ACS (PC = 0.014, OR = 1.37 and PC = 0.006, OR = 1.90, respectively). Based on Hosmer–Lemeshow Goodness of Fit test, the recessive model was selected to estimate risk between ACS patients and controls adjusted by cardiovascular risk factors using a multiple logistic analysis. In this case, the OR adjusted was 1.78 for the 1614 CC genotype (P = 0.023). The analysis of linkage disequilibrium showed one risk haplotype (ACG) and one protective haplotype (AGG) for developing ACS (P = 0.02, OR = 1.5 and P = 0.04, OR = 0.72, respectively). Conclusion: The results suggest that MHC2TA 1614 gene polymorphism could be involved in the risk of developing ACS. © 2013 Elsevier Ltd. All rights reserved.
1. Introduction Coronary artery disease (CAD) is a complex multifactorial and polygenic disorder resulting from an excessive inflammatory response to various forms of injurious stimuli to the arterial wall (Ross, 1999; Garcia-Moll, 2005; Lusis, 2000). Inflammatory processes, genetic background, and various environmental factors, coupled with dyslipidemia, hypertension, diabetes, obesity, alcohol consumption and smoking, play an important role in the progression of the atherosclerotic plaque (Lusis, 2000; Libby, 2002; Virmani et al., 2005). The transition of a stable coronary atherosclerotic lesion into a ruptured and/or eroded plaque results in the clinical manifestation of an acute coronary syndrome (ACS) (Heeschen
∗ Corresponding author at: Department of Molecular Biology, Instituto Nacional de Cardiología “Ignacio Chávez”, Juan Badiano No. 1, Tlalpan 14080, Mexico D.F., Mexico. Tel.: +52 55 5573 2911×1460; fax: +52 55 5573 0926. E-mail address: [email protected] (J.M. Fragoso). 0161-5890/$ – see front matter © 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.molimm.2013.02.007
et al., 2003; Achar et al., 2005). The ACS (unstable angina or myocardial infarction) is caused mainly by rupture or erosion of the atherosclerotic plaque, with subsequent thrombus formation (Achar et al., 2005; Mouco et al., 2006; Carter, 2005). The major histocompatibility complex class II transactivator (MHC2TA) is considered an important molecule in the inflammatory process regulation. The MHC2TA is a transcriptional co-activator, it is the key intermediate responsible for INF-␥-inducible and is required for expression of MHC class II and other genes related to antigen presentation in antigen-presenting cells (Serrat et al., 2010; Patel et al., 2005; Holling et al., 2007; Kim et al., 2006). On the other hand, the MHC2TA is a master regulator of MHC class II transcription and has an important role in the activation of several genes (Xu et al., 2008; Nozell et al., 2004; Zhou et al., 2007; Yee et al., 2004, 2005; LeibundGut-Landmann et al., 2004) that play a role in the development of the atherosclerosis plaque. The MHC2TA gene is located in the 16p13 region (Serrat et al., 2010) and presents several polymorphic sites. Three single nucleotide polymorphisms (SNPs) have been studied more, one in the promoter region −168
G. Vargas-Alarcón et al. / Molecular Immunology 55 (2013) 424–428
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Table 1 Baseline clinical characteristics of the studied individuals. Clinical characteristics
ACS patients (n (%))
Healthy controls (n (%))
P-value
Mena High blood pressure (mmHg) Type II diabetes mellitusa Dyslipidemiaa Smokinga
238 (80) 203 (68) 119 (40) 179 (60) 141 (47) Median (percentile 25–75) 59 (52–66) 26.6 (24.2–29.4)
202 (71) 94 (33) 39 (14) 119 (42) 59 (21) Median (percentile 25–75) 55 (50–61) 27.5 (25.5–29.9)
0.014 <0.001 <0.001 <0.001 <0.001
Age (years) BMI (kg/m2 ) a
<0.001 0.0005
(n (%)) number and proportion of subjects with the clinical characteristic in both groups.
A>G (rs3087456) and two polymorphic sites in the exon 11 at positions 1614 C>G (rs4774), which results in an Ala>Gly substitution at amino acid 500, and 2536 G>A (rs2229320), which results in a silent mutation (Pro>Pro) at amino acid 798. These polymorphisms have been associated with low and abnormal expression of the MHC2TA in systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, myocardial infarction, metabolic syndrome, and atherosclerosis (Sanchez et al., 2008; Koizumi et al., 2006; Swanberg et al., 2005; Lindholm et al., 2006). Considering the prominent role of the MHC2TA as regulator of several genes, this study was based on the assumption that MHC2TA gene polymorphisms have a measurable influence on the development of the atherosclerotic plaque and contribute to or increase the occurrence of ACS. The objective of this study was to establish the role of MHC2TA gene polymorphisms in the risk of developing ACS in a group of Mexicans patients. 2. Materials and methods 2.1. Patients and controls The study included 297 Mexican Mestizo patients with ACS (238 men and 59 women, mean age 59.64 ± 11.31) who were referred to the Instituto Nacional de Cardiología Ignacio Chávez. Twohundred-twelve of them presented myocardial infarction and 85 presented unstable angina. ACS was diagnosed on the basis of clinical history, physical examination with electrocardiography, chest radiography, echocardiography and coronary angiography. The diagnosis of ACS was made according to the World Health Organization and the American Heart Associated (AHA)/American College of Cardiology (ACC) (Richardson et al., 1996). Also, a group of 283 healthy unrelated individuals (mean age 55.4 ± 7.24) with neither symptoms nor previous diagnosis of cardiovascular problems and systemic disease was studied as control group. All included subjects were ethnically matched and we considered as Mexican Mestizos only those individuals who for three generations, including their own, have been born in Mexico. The Institutional Ethics and Research Committees approved the study, and all subjects signed informed consent. 2.2. DNA extraction Genomic DNA from whole blood containing EDTA was isolated by standard technique (Lahiri and Numberger, 1991).
2.4. Statistical analysis Gene frequencies of MHC2TA polymorphisms on patients and controls were obtained by direct counting. The Hardy–Weinberg equilibrium was evaluated by chi-square test. Statistical analysis was carried out with the Stata 10.0 software for Windows. Statistical power to detect association with ACS was 0.80 as estimated with QUANTO software (http://hydra.usc.edu/GxE/). In the exploratory analysis, numerical data showed a different distribution from normal standard (Gaussian) distribution (Shapiro Wilk’s test of normality, P > 0.05); nominal and categorical variables were tabulated, and its proportions estimated. Hypertension, altered lipid profile, smoking, and type 2 diabetes mellitus, each one had two categories, yes or no, and all of them described the current condition of cases and controls. Comparison of numerical variables, such as age and body mass index, between both groups was carried out with Mann Whitney U-test, data are presented as median (25−75th percentile). Categorical variables were analyzed with Chi2 tests or Fisher exact test as needed and presented as absolute frequencies and proportions. The P-values were corrected (PC) according to the number of specificities tested and the number of comparisons performed, and they were considered statistically significant if their value were <0.05. Inheritance hypothesis was tested according to four models: co-dominant, dominant, recessive, and heterozygous advantage, recessive model was selected based on Akaike information criteria. Homozygous CC genotype was used as referential. Multiple logistic models were constructed in order to identify the variables that explain better the risk between cases and controls. Models were constructed including one variable at a time to identify confounding bias; changes in estimated odds ratios (ORs) were less than 10%. In the multiple model, a likelihood-ratio test after estimation was done, no significant changes were identified between the two models in relation to age and BMI. When a principal effect model was reached, effect modification was also tested, interaction and terms were constructed between type 2 diabetes mellitus against lipid profile and hypertension, and gender against smoking habit and polymorphisms, and hypertension against smoking habit, and no significant ORs (P-value ≤ 0.20) were identified and therefore none of these terms were included in the final model. Hosmer–Lemeshow Goodness of fit test was performed for each multiple logistic model. Pairwise linkage disequilibrium (LD, D ) estimations between polymorphisms and haplotype reconstruction were performed with Haploview version 4:1 (Broad Institute of Massachusetts Institute of Technology and Harvard University, Cambridge, MA, USA).
2.3. Determination of the MHC2TA polymorphisms 3. Results The MHC2TA −168 A>G (rs3087456), 1614 C>G (rs4774), and 2536 G>A (rs2229320) single nucleotide polymorphisms were genotyped using 5 exonuclease TaqMan genotyping assays on an ABI Prism 7900 HT Fast Real time PCR System, according to manufacturer’s instructions (Applied Biosystems, Foster City, CA, USA).
3.1. Characteristics of the study sample Baseline characteristics of the ACS patients and healthy controls included in the study are presented in Table 1.
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G. Vargas-Alarcón et al. / Molecular Immunology 55 (2013) 424–428
Table 2 Allele and genotype distribution of MHC2TA gene polymorphisms in ACS patients and healthy controls. ACS n = 297 (n (%)) MHC2TA −168 A/G (rs3087456) Allele 380 (64) G A 214 (36) Genotype 126 (42) GG 128 (43) GA 43 (15) AA MHC2TA C1614G (rs4774) Allele 306 (52) G 288 (48) C Genotype 83 (28) GG 140 (47) GC 74 (25) CC MHC2TA A2536G (rs2229320) Allele 499 (84) G 95 (16) A Genotype 212 (72) GG 75 (25) GA 10 (3) AA
Controls n = 283 (n (%))
PC value
OR
95%IC
369 (65) 197 (35)
0.66 0.66
0.95 1.05
0.73–1.20 1.02–1.35
115 (40) 139 (49) 29 (10)
0.66 0.14 0.12
1.08 0.78 1.48
0.76–1.52 0.56–1.10 0.87–2.53
336 (59) 230 (41)
0.014 0.014
0.73 1.37
0.57–0.92 1.08–1.75
95 (33) 146 (52) 42 (15)
0.14 0.28 0.006
0.77 1.15 1.90
0.53–1.11 0.60–1.17 1.23–2.96
481 (85) 85 (15)
0.64 0.64
0.93 1.05
0.64–1.36 0.75–1.55
206 (73) 69 (24) 8 (3)
0.70 0.80 0.70
0.93 1.05 1.20
0.64–1.36 0.75–1.55 0.43–3.38
Abbrevations: OR, odds ratio; 95%CI, 95% confidence intervals; NS, not significant; PC, P corrected. The significant p values are in bold.
Table 3 Risk analysis (MCH2TA C1614G polymorphism) as function of the inheritance model in ACS and healthy controls. Model
Genotype
OR
Co-dominant
GG CG CC
1 1.10 2.02
0.75–1.60 1.25–3.26
NS 0.012
Dominant
GG CG + CC
1 1.30
0.91–1.86
NS
Recessive
GG + CG CC
1 1.90
1.25–2.90
0.006
CG GG + CC
1 1.19
0.86–1.66
NS
Heterozygous
95% CI
OR (95%IC); P-valuea
PC value
1.78 (1.08–2.93); 0.023
OR, odds ratio; 95%CI, 95% confidence intervals; NS, not significant; PC, P corrected. a The recessive model was adjusted by gender, age, hypertension, dyslipidemia, smoking, diabetes and body mass index.
3.2. Allele and genotype frequencies Allele and genotype frequencies of MHC2TA polymorphisms in ACS patients and healthy controls are shown in Table 2. Observed and expected frequencies of the three polymorphism were in Hardy–Weinberg equilibrium. The distribution of the MHC2TA −168 and MHC2TA 2536 polymorphisms was similar between ACS patients and healthy controls. On the other hand, the frequency of C allele and CC genotype in patients with ACS was larger than in healthy controls (PC = 0.014, OR = 1.37, 95%CI = 1.08–1.75 and PC = 0.006, OR = 1.90, 95%CI = 1.23–2.96, respectively). The inheritance hypothesis for this polymorphism was tested according to four models: co-dominant, dominant, recessive, and heterozygous advantage (Table 3). Based on Hosmer–Lemeshow Goodness of Fit test, the recessive model was selected to estimate risk between ACS patients and controls adjusted by cardiovascular risk factors (gender, age, hypertension, dyslipidemia, smoking, diabetes and body mass index) using a multiple logistic analysis. In this case, the adjusted OR was 1.78 for the CC genotype (P = 0.023, 95%CI = 1.08–2.93) (Table 3). The analysis of the linkage disequilibrium showed two of seven allelic combinations (AGG and ACG) with important differences between the two groups (Table 4). The ACG was considered a risk
haplotype and the AGG a protective haplotype for developing ACS (P = 0.02, OR = 1.50, 95% CI = 1.04–2.15 and P = 0.04, OR = 0.72, 95% CI = 0.52–1.01). The P-values did not remain significant when corrected for multiple comparisons The order of the polymorphisms in the haplotypes corresponds to the positions in the chromosome (MHC2TA −168 A>G (rs3087456), 1614 C>G (rs4774), and 2536 G>A (rs2229320)). Table 4 Frequencies (%) of MHC2TA haplotypes (MHC2TA −168 A>G, 1614 C>G and 2536 G>A) in ACS patients and healthy controls.
Haplotype GCG GGG AGG ACG GGA AGA ACA
ACS (n = 297)
Controls (n = 283)
P
OR
Hf 0.303 0.252 0.131 0.154 0.068 0.064 0.016
Hf 0.290 0.278 0.174 0.108 0.079 0.063 0.006
0.62 0.33 0.04 0.02 0.49 0.97 0.06
1.07 0.88 0.72 1.50 0.86 1.01 3.21
95%CI 0.82–1.38 0.67–1.15 0.52–1.01 1.04–2.15 0.54–1.37 0.61–1.65 0.81–14.8
The order of the polymorphisms in the haplotypes is according to the positions in the chromosome (−168, 1614, 2536). Hf, haplotype frequency; ACS, acute coronary syndrome; OR, odds ratio; CI, confidence interval; P, P-value. The significant p values are in bold.
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4. Discussion The process of the atherosclerotic plaque is characterized by the immune cell infiltrate, typical of chronic inflammation, involving macrophages, lymphocytes, and plasma cells, which allow the formation of the atherosclerotic plaque (Achar et al., 2005; Mouco et al., 2006). Recent studies provide evidence on the emerging role of the MHC2TA gene as master regulator of MHC class II transcription and several genes, such as collagen (Xu et al., 2008), matrix metalloproteinase-9 (Nozell et al., 2004), IL-4 (Zhou et al., 2007), cathepsin E, IL-10, and TGF- (Yee et al., 2004, 2005; LeibundGutLandmann et al., 2004), that play a role in the development of the atherosclerotic plaque. In the present work, we studied three single nucleotide polymorphisms [−168 A>G (rs3087456), 1614 C>G (rs4774), and 2536 G>A (rs2229320)] located in the MHC2TA gene in patients with ACS. In humans, these three polymorphisms have been associated with low and abnormal expression of MHC2TA gene, in particular the −168 G and 1614 C alleles (Sanchez et al., 2008; Koizumi et al., 2006; Swanberg et al., 2005; Lindholm et al., 2006; Orozco et al., 2006; Eyre et al., 2006; Martinez et al., 2007). In addition, several studies associate this gene with high risk of developing complex diseases with inflammatory component, such as systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, myocardial infarction, metabolic syndrome, as well as with cardiovascular mortality (Sanchez et al., 2008; Koizumi et al., 2006; Swanberg et al., 2005; Lindholm et al., 2006; Orozco et al., 2006; Eyre et al., 2006; Martinez et al., 2007). Interestingly, in our study, the MHC2TA 1614 C>G (rs4777) polymorphism was associated with the risk of developing ACS. However, data in the literature that associate this polymorphism with some pathologies are few and controversial. For example, Swanberg et al., did not detected an association of the MHC2TA 1614 C>G polymorphism with the risk of rheumatoid arthritis, multiple sclerosis, and myocardial infarction in their European populations (Swanberg et al., 2005). Also, Koizumi et al., failed to detect an association of systemic lupus erythematosus with either the −168 A>G or the 1614 C>G polymorphism of the MHC2TA gene in a Japanese population (Koizumi et al., 2006). Nonetheless, in recent studies, the MHC2TA 1614 C allele has been associated with several conditions in other populations. Bronson et al., reported that individuals with the 1614 C allele had a 1.20-fold increased risk of developing systemic lupus erythematosus in a Caucasian American population (Bronson et al., 2011). Moreover, Alvarez-Lafuente et al., also reported that the 1614 C allele was significantly increased in Spanish patients with human herpesvirus 6A (HHV6A) (AlvarezLafuente et al., 2010). In addition, Bronson et al. also reported that the 1614 C allele was associated with an increased risk of developing multiple sclerosis (OR = 1.19) in a Caucasian American population (Bronson et al., 2010). The minor allele frequency in these studies ranged from 0.26 to 0.33, whereas in our study was 0.48 in patients and 0.41 in controls. On the other hand, in our study, the haplotype analysis showed strong linkage disequilibrium among the three MHC2TA gene polymorphisms. We detected one haplotype (ACG) of risk and one protective haplotype (AGG) for developing ACS. However this result should be taken with care because the association did not remain significant when P-values were corrected for multiple comparisons. Martinez et al. studied two (−168 A/G and 1614 C/G) MHC2TA polymorphisms and reported the association of the “GC” haplotype with risk of developing multiple sclerosis (Martinez et al., 2007). This haplotype includes the 1614 C allele reported in our risk haplotypes (ACG). The authors suggest that this haplotype could explain the reduced expression of the MHCII gene after inflammatory stimuli. On the other hand, Martinez et al., confirmed that the “GC” haplotype also increases the risk of developing rheumatoid arthritis (Martinez et al., 2010). In their study, authors confirmed that this haplotype is associated with the risk of
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developing complex diseases. Our results and those reported in the literature suggest an important role of the 1614 C/G polymorphism in the genetic susceptibility to several diseases. This polymorphism is located at nucleotide 1614 C>G and causes a conservative substitution of alanine to glycine at amino acid 500. In functional studies, the substitution in the 1614 nucleotide (C>G) was also observed accompanying disease-causing deletion in the MHC2TA cDNA isolated from patients with bare lymphocyte syndrome (Steimle et al., 1993; Reley et al., 1995), a severe immunodeficiency condition that can result from deficient or abnormal expression of MHC2TA (Mach et al., 1996). This suggests that the MHC2TA 1614 G/C polymorphism could be related with mRNA expression. Finally, these findings are in line with our results in which the risk haplotype presents the “C” allele, thus, the individuals with these haplotype could produce less MHC2TA, decreasing the functional effect as master regulator of MHC class II transcription in several genes. In summary, our data suggest that the MHC2TA 1614 C/G polymorphism plays an important role in the risk of developing ACS, with the highest risk for the 1614 C allele and CC genotype. In our study, it was possible to distinguish one risk and one protective haplotype for developing ACS. Additional studies in other populations could help to define the true role of this marker as risk factor for developing ACS. Funding sources This work was supported in part by grants from the Consejo Nacional de Ciencia y Tecnología (project 156911) and Fundación Gonzalo Rio Arronte, Mexico City, Mexico. Conflict of interest No competing financial interests exist. Acknowledgments The authors are grateful to the study participants. Institutional Review Board approval was obtained for all sample collections. References Achar, S.A., Kundu, S., Norcross, W.A., 2005. Diagnosis of acute coronary syndrome. American Family Physician 72, 119–126. Alvarez-Lafuente, R., Martinez, A., Garcia-Montojo, M., et al., 2010. MHC2TA rs4774C and HHV-6a active replication in multiple sclerosis patients. European Journal of Neurology 17, 129–135. Bronson, P.G., Cailler, S., Ramsay, P.P., et al., 2010. CIITA variation in the presence of HLA-DRB1*1501 increases risk for multiple sclerosis. Human Molecular Genetics 19, 2331–2340. Bronson, P.G., Goldstein, B.A., Ramsay, P.P., et al., 2011. The rs4774 CIITA missense variant is associated with risk of systemic lupus erythematosus. Genes and Immunity 12, 667–671. Carter, A.M., 2005. Inflammation thrombosis and acute coronary syndromes. Diabetes & Vascular Disease Research 2, 113–123. Eyre, S., Bowes, J., Spreckley, K., et al., 2006. Investigation of the MHC2TA gene, associated with rheumatoid arthritis in a Swedish population, in a UK rheumatoid arthritis cohort. Arthritis and Rheumatism 54, 3417–3422. Garcia-Moll, X., 2005. Inflammatory and anti-inflammatory markers in acute coronary syndromes. Ready for use in the clinical setting? Revista espanola de cardiologia 58, 615–617. Heeschen, C., Dimmeler, S., Hamm, C.W., et al., 2003. Serum level of the antiinflammatory cytokine interleukin-10 is an important prognostic determinant in patients with acute coronary syndromes. Circulation 107, 2109–2114. Holling, T.M., Bergevoet, M.W., Wilson, L., et al., 2007. A role for EZH2 in silencing of IFN-gamma inducible MHC2TA transcription in uveal melanoma. Journal of Immunology 179, 5317–5325. Kim, T.W., Park, H.J., Choi, E.Y., Jung, K.C., 2006. Overexpression of CIITA in T cells aggravates Th2-mediated colitis in mice. Journal of Korean Medical Science 21, 877–882. Koizumi, K., Okamoto, H., Likuni, N., et al., 2006. Single nucleotide polymorphisms in the gene encoding the major histocompatibility complex class II transactivator (CIITA) in systemic lupus erythematous. Annals of the Rheumatic Diseases 64, 947–950.
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Molecular Immunology journal homepage: www.elsevier.com/locate/molimm
Short communication
The MHC2TA 1614 C>G gene polymorphism is associated with risk of developing acute coronary syndrome b ˜ Gilberto Vargas-Alarcón a , Marco Antonio Martínez-Ríos b , Marco Antonio Pena-Duque , c c d ˜ , Maite Vallejo , Julián Ramírez-Bello e , Carlos Posadas-Romero , Guillermo Cardoso-Saldana Oscar Pérez-Méndez a , José Manuel Fragoso a,∗ a
Department of Molecular Biology, Instituto Nacional de Cardiología Ignacio Chávez, Mexico City, Mexico Department of Interventional Cardiology, Instituto Nacional de Cardiología Ignacio Chávez, Mexico City, Mexico c Department of Endocrinology, Instituto Nacional de Cardiología Ignacio Chávez, Mexico City, Mexico d Department of Sociomedicine, Instituto Nacional de Cardiología Ignacio Chávez, Mexico City, Mexico e Immunogenomics and Metabolic Diseases Laboratory, Instituto Nacional de Medicina Genómica, Mexico City, Mexico b
a r t i c l e
i n f o
Article history: Received 11 February 2013 Received in revised form 21 February 2013 Accepted 25 February 2013 Available online 16 March 2013 Keywords: Acute coronary syndrome Genetic susceptibility Histocompatibility complex class II trans-activator (MHC2TA) Polymorphism
a b s t r a c t Background: Inflammation plays an essential role in the development and progression of atherosclerotic lesions. The major histocompatibility complex class II trans-activator (MHC2TA) is considered an important molecule in the inflammatory process regulation. The aim of the present study was to evaluate the role of MHC2TA gene polymorphisms as susceptibility markers for acute coronary syndrome (ACS). Methods: Three polymorphisms (−168 A>G, 1614 C>G, and 2536 G>A) of the MHC2TA gene were analyzed by 5 exonuclease TaqMan genotyping assays in a group of 297 patients with ACS and 283 healthy controls. Haplotypes were constructed after linkage disequilibrium analysis. Results: The 1614 C allele and CC genotype were associated with risk of developing ACS (PC = 0.014, OR = 1.37 and PC = 0.006, OR = 1.90, respectively). Based on Hosmer–Lemeshow Goodness of Fit test, the recessive model was selected to estimate risk between ACS patients and controls adjusted by cardiovascular risk factors using a multiple logistic analysis. In this case, the OR adjusted was 1.78 for the 1614 CC genotype (P = 0.023). The analysis of linkage disequilibrium showed one risk haplotype (ACG) and one protective haplotype (AGG) for developing ACS (P = 0.02, OR = 1.5 and P = 0.04, OR = 0.72, respectively). Conclusion: The results suggest that MHC2TA 1614 gene polymorphism could be involved in the risk of developing ACS. © 2013 Elsevier Ltd. All rights reserved.
1. Introduction Coronary artery disease (CAD) is a complex multifactorial and polygenic disorder resulting from an excessive inflammatory response to various forms of injurious stimuli to the arterial wall (Ross, 1999; Garcia-Moll, 2005; Lusis, 2000). Inflammatory processes, genetic background, and various environmental factors, coupled with dyslipidemia, hypertension, diabetes, obesity, alcohol consumption and smoking, play an important role in the progression of the atherosclerotic plaque (Lusis, 2000; Libby, 2002; Virmani et al., 2005). The transition of a stable coronary atherosclerotic lesion into a ruptured and/or eroded plaque results in the clinical manifestation of an acute coronary syndrome (ACS) (Heeschen
∗ Corresponding author at: Department of Molecular Biology, Instituto Nacional de Cardiología “Ignacio Chávez”, Juan Badiano No. 1, Tlalpan 14080, Mexico D.F., Mexico. Tel.: +52 55 5573 2911×1460; fax: +52 55 5573 0926. E-mail address: [email protected] (J.M. Fragoso). 0161-5890/$ – see front matter © 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.molimm.2013.02.007
et al., 2003; Achar et al., 2005). The ACS (unstable angina or myocardial infarction) is caused mainly by rupture or erosion of the atherosclerotic plaque, with subsequent thrombus formation (Achar et al., 2005; Mouco et al., 2006; Carter, 2005). The major histocompatibility complex class II transactivator (MHC2TA) is considered an important molecule in the inflammatory process regulation. The MHC2TA is a transcriptional co-activator, it is the key intermediate responsible for INF-␥-inducible and is required for expression of MHC class II and other genes related to antigen presentation in antigen-presenting cells (Serrat et al., 2010; Patel et al., 2005; Holling et al., 2007; Kim et al., 2006). On the other hand, the MHC2TA is a master regulator of MHC class II transcription and has an important role in the activation of several genes (Xu et al., 2008; Nozell et al., 2004; Zhou et al., 2007; Yee et al., 2004, 2005; LeibundGut-Landmann et al., 2004) that play a role in the development of the atherosclerosis plaque. The MHC2TA gene is located in the 16p13 region (Serrat et al., 2010) and presents several polymorphic sites. Three single nucleotide polymorphisms (SNPs) have been studied more, one in the promoter region −168
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Table 1 Baseline clinical characteristics of the studied individuals. Clinical characteristics
ACS patients (n (%))
Healthy controls (n (%))
P-value
Mena High blood pressure (mmHg) Type II diabetes mellitusa Dyslipidemiaa Smokinga
238 (80) 203 (68) 119 (40) 179 (60) 141 (47) Median (percentile 25–75) 59 (52–66) 26.6 (24.2–29.4)
202 (71) 94 (33) 39 (14) 119 (42) 59 (21) Median (percentile 25–75) 55 (50–61) 27.5 (25.5–29.9)
0.014 <0.001 <0.001 <0.001 <0.001
Age (years) BMI (kg/m2 ) a
<0.001 0.0005
(n (%)) number and proportion of subjects with the clinical characteristic in both groups.
A>G (rs3087456) and two polymorphic sites in the exon 11 at positions 1614 C>G (rs4774), which results in an Ala>Gly substitution at amino acid 500, and 2536 G>A (rs2229320), which results in a silent mutation (Pro>Pro) at amino acid 798. These polymorphisms have been associated with low and abnormal expression of the MHC2TA in systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, myocardial infarction, metabolic syndrome, and atherosclerosis (Sanchez et al., 2008; Koizumi et al., 2006; Swanberg et al., 2005; Lindholm et al., 2006). Considering the prominent role of the MHC2TA as regulator of several genes, this study was based on the assumption that MHC2TA gene polymorphisms have a measurable influence on the development of the atherosclerotic plaque and contribute to or increase the occurrence of ACS. The objective of this study was to establish the role of MHC2TA gene polymorphisms in the risk of developing ACS in a group of Mexicans patients. 2. Materials and methods 2.1. Patients and controls The study included 297 Mexican Mestizo patients with ACS (238 men and 59 women, mean age 59.64 ± 11.31) who were referred to the Instituto Nacional de Cardiología Ignacio Chávez. Twohundred-twelve of them presented myocardial infarction and 85 presented unstable angina. ACS was diagnosed on the basis of clinical history, physical examination with electrocardiography, chest radiography, echocardiography and coronary angiography. The diagnosis of ACS was made according to the World Health Organization and the American Heart Associated (AHA)/American College of Cardiology (ACC) (Richardson et al., 1996). Also, a group of 283 healthy unrelated individuals (mean age 55.4 ± 7.24) with neither symptoms nor previous diagnosis of cardiovascular problems and systemic disease was studied as control group. All included subjects were ethnically matched and we considered as Mexican Mestizos only those individuals who for three generations, including their own, have been born in Mexico. The Institutional Ethics and Research Committees approved the study, and all subjects signed informed consent. 2.2. DNA extraction Genomic DNA from whole blood containing EDTA was isolated by standard technique (Lahiri and Numberger, 1991).
2.4. Statistical analysis Gene frequencies of MHC2TA polymorphisms on patients and controls were obtained by direct counting. The Hardy–Weinberg equilibrium was evaluated by chi-square test. Statistical analysis was carried out with the Stata 10.0 software for Windows. Statistical power to detect association with ACS was 0.80 as estimated with QUANTO software (http://hydra.usc.edu/GxE/). In the exploratory analysis, numerical data showed a different distribution from normal standard (Gaussian) distribution (Shapiro Wilk’s test of normality, P > 0.05); nominal and categorical variables were tabulated, and its proportions estimated. Hypertension, altered lipid profile, smoking, and type 2 diabetes mellitus, each one had two categories, yes or no, and all of them described the current condition of cases and controls. Comparison of numerical variables, such as age and body mass index, between both groups was carried out with Mann Whitney U-test, data are presented as median (25−75th percentile). Categorical variables were analyzed with Chi2 tests or Fisher exact test as needed and presented as absolute frequencies and proportions. The P-values were corrected (PC) according to the number of specificities tested and the number of comparisons performed, and they were considered statistically significant if their value were <0.05. Inheritance hypothesis was tested according to four models: co-dominant, dominant, recessive, and heterozygous advantage, recessive model was selected based on Akaike information criteria. Homozygous CC genotype was used as referential. Multiple logistic models were constructed in order to identify the variables that explain better the risk between cases and controls. Models were constructed including one variable at a time to identify confounding bias; changes in estimated odds ratios (ORs) were less than 10%. In the multiple model, a likelihood-ratio test after estimation was done, no significant changes were identified between the two models in relation to age and BMI. When a principal effect model was reached, effect modification was also tested, interaction and terms were constructed between type 2 diabetes mellitus against lipid profile and hypertension, and gender against smoking habit and polymorphisms, and hypertension against smoking habit, and no significant ORs (P-value ≤ 0.20) were identified and therefore none of these terms were included in the final model. Hosmer–Lemeshow Goodness of fit test was performed for each multiple logistic model. Pairwise linkage disequilibrium (LD, D ) estimations between polymorphisms and haplotype reconstruction were performed with Haploview version 4:1 (Broad Institute of Massachusetts Institute of Technology and Harvard University, Cambridge, MA, USA).
2.3. Determination of the MHC2TA polymorphisms 3. Results The MHC2TA −168 A>G (rs3087456), 1614 C>G (rs4774), and 2536 G>A (rs2229320) single nucleotide polymorphisms were genotyped using 5 exonuclease TaqMan genotyping assays on an ABI Prism 7900 HT Fast Real time PCR System, according to manufacturer’s instructions (Applied Biosystems, Foster City, CA, USA).
3.1. Characteristics of the study sample Baseline characteristics of the ACS patients and healthy controls included in the study are presented in Table 1.
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Table 2 Allele and genotype distribution of MHC2TA gene polymorphisms in ACS patients and healthy controls. ACS n = 297 (n (%)) MHC2TA −168 A/G (rs3087456) Allele 380 (64) G A 214 (36) Genotype 126 (42) GG 128 (43) GA 43 (15) AA MHC2TA C1614G (rs4774) Allele 306 (52) G 288 (48) C Genotype 83 (28) GG 140 (47) GC 74 (25) CC MHC2TA A2536G (rs2229320) Allele 499 (84) G 95 (16) A Genotype 212 (72) GG 75 (25) GA 10 (3) AA
Controls n = 283 (n (%))
PC value
OR
95%IC
369 (65) 197 (35)
0.66 0.66
0.95 1.05
0.73–1.20 1.02–1.35
115 (40) 139 (49) 29 (10)
0.66 0.14 0.12
1.08 0.78 1.48
0.76–1.52 0.56–1.10 0.87–2.53
336 (59) 230 (41)
0.014 0.014
0.73 1.37
0.57–0.92 1.08–1.75
95 (33) 146 (52) 42 (15)
0.14 0.28 0.006
0.77 1.15 1.90
0.53–1.11 0.60–1.17 1.23–2.96
481 (85) 85 (15)
0.64 0.64
0.93 1.05
0.64–1.36 0.75–1.55
206 (73) 69 (24) 8 (3)
0.70 0.80 0.70
0.93 1.05 1.20
0.64–1.36 0.75–1.55 0.43–3.38
Abbrevations: OR, odds ratio; 95%CI, 95% confidence intervals; NS, not significant; PC, P corrected. The significant p values are in bold.
Table 3 Risk analysis (MCH2TA C1614G polymorphism) as function of the inheritance model in ACS and healthy controls. Model
Genotype
OR
Co-dominant
GG CG CC
1 1.10 2.02
0.75–1.60 1.25–3.26
NS 0.012
Dominant
GG CG + CC
1 1.30
0.91–1.86
NS
Recessive
GG + CG CC
1 1.90
1.25–2.90
0.006
CG GG + CC
1 1.19
0.86–1.66
NS
Heterozygous
95% CI
OR (95%IC); P-valuea
PC value
1.78 (1.08–2.93); 0.023
OR, odds ratio; 95%CI, 95% confidence intervals; NS, not significant; PC, P corrected. a The recessive model was adjusted by gender, age, hypertension, dyslipidemia, smoking, diabetes and body mass index.
3.2. Allele and genotype frequencies Allele and genotype frequencies of MHC2TA polymorphisms in ACS patients and healthy controls are shown in Table 2. Observed and expected frequencies of the three polymorphism were in Hardy–Weinberg equilibrium. The distribution of the MHC2TA −168 and MHC2TA 2536 polymorphisms was similar between ACS patients and healthy controls. On the other hand, the frequency of C allele and CC genotype in patients with ACS was larger than in healthy controls (PC = 0.014, OR = 1.37, 95%CI = 1.08–1.75 and PC = 0.006, OR = 1.90, 95%CI = 1.23–2.96, respectively). The inheritance hypothesis for this polymorphism was tested according to four models: co-dominant, dominant, recessive, and heterozygous advantage (Table 3). Based on Hosmer–Lemeshow Goodness of Fit test, the recessive model was selected to estimate risk between ACS patients and controls adjusted by cardiovascular risk factors (gender, age, hypertension, dyslipidemia, smoking, diabetes and body mass index) using a multiple logistic analysis. In this case, the adjusted OR was 1.78 for the CC genotype (P = 0.023, 95%CI = 1.08–2.93) (Table 3). The analysis of the linkage disequilibrium showed two of seven allelic combinations (AGG and ACG) with important differences between the two groups (Table 4). The ACG was considered a risk
haplotype and the AGG a protective haplotype for developing ACS (P = 0.02, OR = 1.50, 95% CI = 1.04–2.15 and P = 0.04, OR = 0.72, 95% CI = 0.52–1.01). The P-values did not remain significant when corrected for multiple comparisons The order of the polymorphisms in the haplotypes corresponds to the positions in the chromosome (MHC2TA −168 A>G (rs3087456), 1614 C>G (rs4774), and 2536 G>A (rs2229320)). Table 4 Frequencies (%) of MHC2TA haplotypes (MHC2TA −168 A>G, 1614 C>G and 2536 G>A) in ACS patients and healthy controls.
Haplotype GCG GGG AGG ACG GGA AGA ACA
ACS (n = 297)
Controls (n = 283)
P
OR
Hf 0.303 0.252 0.131 0.154 0.068 0.064 0.016
Hf 0.290 0.278 0.174 0.108 0.079 0.063 0.006
0.62 0.33 0.04 0.02 0.49 0.97 0.06
1.07 0.88 0.72 1.50 0.86 1.01 3.21
95%CI 0.82–1.38 0.67–1.15 0.52–1.01 1.04–2.15 0.54–1.37 0.61–1.65 0.81–14.8
The order of the polymorphisms in the haplotypes is according to the positions in the chromosome (−168, 1614, 2536). Hf, haplotype frequency; ACS, acute coronary syndrome; OR, odds ratio; CI, confidence interval; P, P-value. The significant p values are in bold.
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4. Discussion The process of the atherosclerotic plaque is characterized by the immune cell infiltrate, typical of chronic inflammation, involving macrophages, lymphocytes, and plasma cells, which allow the formation of the atherosclerotic plaque (Achar et al., 2005; Mouco et al., 2006). Recent studies provide evidence on the emerging role of the MHC2TA gene as master regulator of MHC class II transcription and several genes, such as collagen (Xu et al., 2008), matrix metalloproteinase-9 (Nozell et al., 2004), IL-4 (Zhou et al., 2007), cathepsin E, IL-10, and TGF- (Yee et al., 2004, 2005; LeibundGutLandmann et al., 2004), that play a role in the development of the atherosclerotic plaque. In the present work, we studied three single nucleotide polymorphisms [−168 A>G (rs3087456), 1614 C>G (rs4774), and 2536 G>A (rs2229320)] located in the MHC2TA gene in patients with ACS. In humans, these three polymorphisms have been associated with low and abnormal expression of MHC2TA gene, in particular the −168 G and 1614 C alleles (Sanchez et al., 2008; Koizumi et al., 2006; Swanberg et al., 2005; Lindholm et al., 2006; Orozco et al., 2006; Eyre et al., 2006; Martinez et al., 2007). In addition, several studies associate this gene with high risk of developing complex diseases with inflammatory component, such as systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, myocardial infarction, metabolic syndrome, as well as with cardiovascular mortality (Sanchez et al., 2008; Koizumi et al., 2006; Swanberg et al., 2005; Lindholm et al., 2006; Orozco et al., 2006; Eyre et al., 2006; Martinez et al., 2007). Interestingly, in our study, the MHC2TA 1614 C>G (rs4777) polymorphism was associated with the risk of developing ACS. However, data in the literature that associate this polymorphism with some pathologies are few and controversial. For example, Swanberg et al., did not detected an association of the MHC2TA 1614 C>G polymorphism with the risk of rheumatoid arthritis, multiple sclerosis, and myocardial infarction in their European populations (Swanberg et al., 2005). Also, Koizumi et al., failed to detect an association of systemic lupus erythematosus with either the −168 A>G or the 1614 C>G polymorphism of the MHC2TA gene in a Japanese population (Koizumi et al., 2006). Nonetheless, in recent studies, the MHC2TA 1614 C allele has been associated with several conditions in other populations. Bronson et al., reported that individuals with the 1614 C allele had a 1.20-fold increased risk of developing systemic lupus erythematosus in a Caucasian American population (Bronson et al., 2011). Moreover, Alvarez-Lafuente et al., also reported that the 1614 C allele was significantly increased in Spanish patients with human herpesvirus 6A (HHV6A) (AlvarezLafuente et al., 2010). In addition, Bronson et al. also reported that the 1614 C allele was associated with an increased risk of developing multiple sclerosis (OR = 1.19) in a Caucasian American population (Bronson et al., 2010). The minor allele frequency in these studies ranged from 0.26 to 0.33, whereas in our study was 0.48 in patients and 0.41 in controls. On the other hand, in our study, the haplotype analysis showed strong linkage disequilibrium among the three MHC2TA gene polymorphisms. We detected one haplotype (ACG) of risk and one protective haplotype (AGG) for developing ACS. However this result should be taken with care because the association did not remain significant when P-values were corrected for multiple comparisons. Martinez et al. studied two (−168 A/G and 1614 C/G) MHC2TA polymorphisms and reported the association of the “GC” haplotype with risk of developing multiple sclerosis (Martinez et al., 2007). This haplotype includes the 1614 C allele reported in our risk haplotypes (ACG). The authors suggest that this haplotype could explain the reduced expression of the MHCII gene after inflammatory stimuli. On the other hand, Martinez et al., confirmed that the “GC” haplotype also increases the risk of developing rheumatoid arthritis (Martinez et al., 2010). In their study, authors confirmed that this haplotype is associated with the risk of
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developing complex diseases. Our results and those reported in the literature suggest an important role of the 1614 C/G polymorphism in the genetic susceptibility to several diseases. This polymorphism is located at nucleotide 1614 C>G and causes a conservative substitution of alanine to glycine at amino acid 500. In functional studies, the substitution in the 1614 nucleotide (C>G) was also observed accompanying disease-causing deletion in the MHC2TA cDNA isolated from patients with bare lymphocyte syndrome (Steimle et al., 1993; Reley et al., 1995), a severe immunodeficiency condition that can result from deficient or abnormal expression of MHC2TA (Mach et al., 1996). This suggests that the MHC2TA 1614 G/C polymorphism could be related with mRNA expression. Finally, these findings are in line with our results in which the risk haplotype presents the “C” allele, thus, the individuals with these haplotype could produce less MHC2TA, decreasing the functional effect as master regulator of MHC class II transcription in several genes. In summary, our data suggest that the MHC2TA 1614 C/G polymorphism plays an important role in the risk of developing ACS, with the highest risk for the 1614 C allele and CC genotype. In our study, it was possible to distinguish one risk and one protective haplotype for developing ACS. Additional studies in other populations could help to define the true role of this marker as risk factor for developing ACS. Funding sources This work was supported in part by grants from the Consejo Nacional de Ciencia y Tecnología (project 156911) and Fundación Gonzalo Rio Arronte, Mexico City, Mexico. Conflict of interest No competing financial interests exist. Acknowledgments The authors are grateful to the study participants. Institutional Review Board approval was obtained for all sample collections. References Achar, S.A., Kundu, S., Norcross, W.A., 2005. Diagnosis of acute coronary syndrome. American Family Physician 72, 119–126. Alvarez-Lafuente, R., Martinez, A., Garcia-Montojo, M., et al., 2010. MHC2TA rs4774C and HHV-6a active replication in multiple sclerosis patients. European Journal of Neurology 17, 129–135. Bronson, P.G., Cailler, S., Ramsay, P.P., et al., 2010. CIITA variation in the presence of HLA-DRB1*1501 increases risk for multiple sclerosis. Human Molecular Genetics 19, 2331–2340. Bronson, P.G., Goldstein, B.A., Ramsay, P.P., et al., 2011. The rs4774 CIITA missense variant is associated with risk of systemic lupus erythematosus. Genes and Immunity 12, 667–671. Carter, A.M., 2005. Inflammation thrombosis and acute coronary syndromes. Diabetes & Vascular Disease Research 2, 113–123. Eyre, S., Bowes, J., Spreckley, K., et al., 2006. Investigation of the MHC2TA gene, associated with rheumatoid arthritis in a Swedish population, in a UK rheumatoid arthritis cohort. Arthritis and Rheumatism 54, 3417–3422. Garcia-Moll, X., 2005. Inflammatory and anti-inflammatory markers in acute coronary syndromes. Ready for use in the clinical setting? Revista espanola de cardiologia 58, 615–617. Heeschen, C., Dimmeler, S., Hamm, C.W., et al., 2003. Serum level of the antiinflammatory cytokine interleukin-10 is an important prognostic determinant in patients with acute coronary syndromes. Circulation 107, 2109–2114. Holling, T.M., Bergevoet, M.W., Wilson, L., et al., 2007. A role for EZH2 in silencing of IFN-gamma inducible MHC2TA transcription in uveal melanoma. Journal of Immunology 179, 5317–5325. Kim, T.W., Park, H.J., Choi, E.Y., Jung, K.C., 2006. Overexpression of CIITA in T cells aggravates Th2-mediated colitis in mice. Journal of Korean Medical Science 21, 877–882. Koizumi, K., Okamoto, H., Likuni, N., et al., 2006. Single nucleotide polymorphisms in the gene encoding the major histocompatibility complex class II transactivator (CIITA) in systemic lupus erythematous. Annals of the Rheumatic Diseases 64, 947–950.
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