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Association of Glucocorticoid Receptor Gene NR3C1 Genetic Variants with Angiographically Documented Coronary Artery Disease and Its Risk Factors
Archives of Medical Research 44 (2013) 27e33
ORIGINAL ARTICLE
Association of Glucocorticoid Receptor Gene NR3C1 Genetic Variants with Angiographically Documented Coronary Artery Disease and Its Risk Factors a _ Iwona Gora˛cy,a Jaros1aw Gora˛cy,b Krzysztof Safranow,c Karolina Skonieczna-Zydecka, and Andrzej Ciechanowicza a
Department of Clinical and Molecular Biochemistry, Pomeranian Medical University, Szczecin, Poland b Clinic of Cardiology, Pomeranian Medical University, Szczecin, Poland c Department of Biochemistry and Medical Chemistry, Pomeranian Medical University, Szczecin, Poland Received for publication June 21, 2012; accepted October 30, 2012 (ARCMED-D-12-00315).
Background and Aims. Glucocorticoids and their receptors are involved in inflammation and many cardiovascular risk factors. We examined associations of Tth111I, N363S and ER22/23EK NR3C1 gene polymorphisms and haplotypes, with coronary artery disease (CAD), severity of CAD (single-vessel vs. multivessel disease) and risk factors. Methods. Three hundred ten individuals were submitted to coronary angiography. Selected genotypes were determined by PCR-RFLP. Results. Carriers of the Tth111I allele T were found significantly less often in the CAD compared with the non-CAD group (49.7 vs. 64.6%, p 5 0.013); this association was similar for TGA haplotype carriers (49.2 vs. 62.8%, p 5 0.024); the T allele was more frequent in females (66.3 vs. 51.1%, p 5 0.020) and its presence was associated with higher levels of HDL-cholesterol (46.6 12.7 vs. 43.5 10.1 mg/dL for T-carriers vs. noncarriers, p 5 0.045). The TT genotype proved to be less common in MVD than SVD (5.9 vs. 14.1%, p 5 0.075). The 363S allele was significantly associated with diabetes mellitus (DM) (24.4 vs. 10.9%; carriers in DM and non-DM subjects, respectively, p 5 0.027), the TT genotype or TGA/TGA diplotype (in which the 363S allele was absent) were less frequent in DM than in non-DM subjects ( p 5 0.012 for Tth111ITT and p 5 0.020 for TGA/TGA diplotype). No significant associations between CAD and N363S or ER22/23EK polymorphisms were found. Conclusions. Our results suggest that the Tth111I NR3C1 polymorphism may play a protective role in the development of CAD, and homozygous TT in development of MVD. The N363S polymorphisms may contribute to the development of diabetes in the Polish population. Ó 2013 IMSS. Published by Elsevier Inc. Key Words: Glucocorticoid receptor, Coronary artery disease, Risk factors, Polymorphism.
Introduction Glucocorticoids (GC) are regulators of the immune system, inflammatory processes, and many other process involved in fat and glucose metabolism (1,2). Inflammatory processes have been recognized to play a key role in the pathogenesis of
Address reprint requests to: Iwona Gora˛cy, Department of Clinical and Molecular Biochemistry, Pomeranian Medical, University, Szczecin, Al. Powsta nc ow Wlkp. 72, 70-111 Szczecin, Poland; Phone: þ48 91 466 14 90; FAX: þ48 91 466 14 92; E-mail: [email protected]
atherosclerosis and coronary artery disease (CAD) (3). It is known that sensitivity to glucocorticoids varies considerably between individuals (4) and several studies have showed that high levels of glucocorticoids result in unfavorable cardiovascular risk factors such as central obesity, steroidinduced diabetes, or dyslipidemia (5e7). The glucocorticoid receptor (GR) is a crucial factor in glucocorticoid responses. GR gene mutations lead to reduced intracellular concentrations or activity of GR in glucocorticoid target tissues (8,9). One previous report described that polymorphisms in the glucocorticoid
0188-4409/$ - see front matter. Copyright Ó 2013 IMSS. Published by Elsevier Inc. http://dx.doi.org/10.1016/j.arcmed.2012.10.020
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Gora˛cy et al./ Archives of Medical Research 44 (2013) 27e33
receptor, NR3C1 gene: ER22/23EK; BcII C/G, N363S; and 9b A/G, modulate glucocorticoid sensitivity (10e12) and two polymorphisms in the NR3C1 gene, the N363S and BcII variants, have been associated with an increased sensitivity to glucocorticoids in vivo; carriers had lower cortisol levels after a dexamethasone suppression test (13,4). Genetic variation in the NR3C1 gene has the potential to predispose to several risk factors for atherosclerosis: higher body mass index (BMI), elevated plasma lipid levels, lower C-reactive protein (CRP) levels, and insulin resistance (4,6). In addition genetic variants in the NR3C1 gene have shown an association with CAD and essential hypertension (HT) (5,7). However, there are disparities in the published literature. In some studies no association was observed (14e16). For example, three polymorphisms (ER22/ 23ER, N363S, and BclI) were studied in a population of elderly persons together with the risk for cardiovascular disease and no association was found (17). In another study of patients with stable CAD and haplotype analysis of the NR3C1 gene, an association was found with self-reported heart failure or systolic dysfunction (18). In a further multicenter study, two common GR gene haplotypes modified cardiovascular disease susceptibility in males (13). Considering these important but contrasting findings, additional studies on the relationship between NR3C1 gene polymorphisms and CAD or its risk factors (diabetes, HT, dyslipidemia) are needed. Therefore, in the present study we assessed the impact of the Tth111I, N363S, ER22/ 23EK polymorphisms of the NR3C1 gene and reconstructed their haplotypes on the prevalence of CAD or its risk factors in a sample of Polish patients subjected to coronary angiography, Associations between the polymorphisms and the extent of coronary vessel occlusion were also studied.
Materials and Methods Study Group The study protocol was approved by the local ethics committee with formal informed consent signed by all the participants. The patients were randomly recruited from the in-patient Cardiology Department, Pomeranian Medical University, Szczecin, Poland, with 310 individuals presenting with a history of CAD or stenocardiac symptoms screened for this study. Of these, 197 patients with $50% occlusion of at least one major coronary artery lumen in angiography comprised the CAD group, regardless of further possible treatment. The group was divided into 78 patients with single-vessel disease (SVD) and 119 with multivessel disease (MVD). The control group (non-CAD) consisted of the remaining 113 subjects without changes in coronary arteries. Patients with a history of myocardial infarction or with clinical diagnosis of cardiomyopathy, coagulopathy, collagenosis and chronic inflammatory disease were
excluded from the study. Arterial hypertension was defined as either systolic blood pressure O140 mmHg or diastolic blood pressure O90 mmHg or antihypertensive treatment; and diabetes mellitus as patients using antidiabetic medication or fasting plasma glucose O6.9 mmol/L. BMI was calculated as mass/height2. Patients were classified as current smokers if they reported a daily rate of more than five cigarettes. Otherwise, patients were classified as nonsmokers. Serum concentrations of total cholesterol (TC), triacylglycerols (TG), high-density lipoprotein cholesterol (HDL-c) and low-density lipoprotein cholesterol (LDL-c) were measured using enzymatic methods (Roche Diagnostics, Warsaw, Poland). Coronary angiography was performed according to standard procedures using Philips INTEGRIS HM 3000 and Philips ALURA (Philips, The Netherlands) devices. Genotyping Genomic DNA was extracted from peripheral blood leukocytes using the QIAamp DNA Mini Kit (QIAGEN, Hilden, Germany). For analysis of the Tth111I [C(-3807)T, rs 10052957)] polymorphism in the NR3C1 gene a polymerase chain reactionerestriction fragment length polymorphism (PCR/RFLP) method was designed with the following primer pair—forward: 5’-TATTTgTTgggTgCCTgCTATgTA-3’ and reverse 5’-gATgAACTCCAgTgTgCCAgAAAg3’ (TIB MOL BIOL, Poznan, Poland). The Tth111I NR3C1amplicons were subsequently digested using the PsyI restriction enzyme (MBI Fermentas, Vilnius, Lithuania). The PCR Tth111I NR3C1 gene product of 367 base pairs (bp) was cut into 278-bp and 89-bp fragments in the presence of C allele and 367 bp remained undigested in case of the T allele. DNA fragments that contained the N363S (A1220G, rs6195) NR3C1 polymorphism were amplified by PCR using primers: forward 5’-ATTCCCgTTggTTCCgAC-3’ and reverse 5’-ATCCCAggTCATTTCCCATC-3’ (TIB MOL BIOL). PCR-RFLP with the MunI restriction enzyme (MBI Fermentas) was performed. In the case of the G allele the final product of 125 bp remained undigested, whereas the A variant gave restriction fragments of 107 bp and 18 bp. For analysis of the ER22/ER23 (G198A/G200A, rs6190) polymorphism of NR3C1, PCR used forward 5’-gATTCggTCATTTCCCATC3’ and reverse 5’-ATCCCAggTCATTTCCCATC-3’ primers. Amplicons were subsequently digested using the MnlI restriction enzyme (MBI Fermentas). The PCR product (442 bp) was cut into fragments of 164 bp, 143 bp for the G200 allele and 178 bp, 164 bp for the A200 allele. Restriction fragments in each case were electrophoretically separated and visualized in ethidium bromide-stained 3% agarose gels. Statistical Analysis Quantitative variables were compared between groups using Kruskal-Wallis or Mann-Whitney tests. c2 or Fisher
Glucocorticoid Receptor Genes and Coronary Artery Disease
29
Table 1. Characteristics of patients with CAD, SVD, advanced disease (MVD) and without CAD (non-CAD) Parameter Male gender Age years BMI (kg/m2) Triacylglycerols (mg/dL) Total cholesterol (mg/dL) HDL-cholesterol (mg/dL) LDL-cholesterol (mg/dL) Smoking Diabetes Hypertension
Non-CAD (n 5 113)
CAD (n 5 197)
p CAD vs. non-CADa
SVD (n 5 78)
MVD (n 5 119)
p MVD vs. SVDa
59.0% 55.2 9.6 26.6 4.1 161.6 88.0 219.6 37.7 49.2 14.0 131.7 29.3 23.0% 8.9% 48.7%
81.7% 57.2 9.1 27.8 3.7 177 70.0 225.9 41.8 43.0 9.4 138.7 33.4 37.6% 17.8% 61.4%
!0.00002 0.082 0.017 0.0068 0.11 0.000055 0.0364 0.008 0.043 0.032
81.0% 54.9 8.0 27.8 3.7 181.8 96.2 222.5 40.6 43.9 9.9 134.9 31.3 36.7% 11.4% 54.4%
82.8% 58.7 9.5 27.8 3.7 174.0 67.3 228.3 42.5 42.4 8.9 141.2 34.6 37.7% 22.1% 65.6%
0.87 0.010 0.80 0.57 0.25 0.26 0.11 0.87 0.014 0.11
CAD, coronary artery disease; SVD, single-vessel disease; MVD, multivessel disease. Data are given as mean SD or %. a p values were calculated with Mann-Whitney test for quantitative variables and with Fisher exact test for qualitative variables.
exact test was used for qualitative variables (genetic data, gender, presence of diabetes, CAD or MVD and smoking status). An exact test was applied to assess conformity of the genotype distribution to Hardy-Weinberg law (19). A multivariate logistic regression model was used to analyze genotypeephenotype association after adjustment for potential confounders (age, gender, BMI, smoking status, presence of diabetes or CAD and HDL-c concentration). Haplotypes were analyzed using the Haploview 4.2 program; p !0.05 was considered statistically significant. Results Baseline characteristics of CAD and non-CAD subjects are shown in Table 1. Differences between patients with SVD and MVD are also presented in this Table 1. Frequency of male gender and value of BMI proved to be significantly higher in the CAD group when compared to controls ( p !0.00002 and p 5 0.017 respectively). Additionally, smoking, diabetes and HT were more common among CAD patients than controls ( p 5 0.008, p 5 0.043 and p 5 0.032, respectively), and serum concentrations of TG and LDL-c were higher among CAD patients than controls ( p 5 0.036 and p 5 0.0068, respectively); HDL-c was lower in CAD patients ( p 5 0.000055). A significant difference in the clinical and biochemical characteristics between patients with SVD and MVD was noted only for age and diabetes (Table 1). The Tth111I, N363S or ER22/23EKeNR3C1 genotypes (Table 2) were found to be in Hardy-Weinberg equilibrium in both CAD and non-CAD groups ( p O0.1). The three loci were then examined for the level of LD in the entire group (n 5 310). The estimated values of D’ were equal to 1 for each pair of loci. Frequencies of haplotypes (Tth111I-ER22/23EK-N363S) were 61.2% for CGA, 31.3% for TGA, 6.5% for CGG and 1.0% for TAA. We observed a significant association between CAD and genotypes of Tth111I (Table 2). Analysis revealed that carriers of the Tth111I allele T (CTþTT genotypes) were significantly less common in the CAD group than in
the non-CAD group (49.7 vs. 64.6%, p 5 0.013). This association was similar for carriers of the TGA haplotype that contain the Tth111I-T allele (49.2 vs. 62.8%, p 5 0.024). However, we also noted that the Tth111I-T allele occurred more frequently in females than in males (66.3 vs. 51.1%; T carriers for combined groups, p 5 0.020) and its presence was associated with a higher level of HDL-c (46.6 12.7 vs. 43.5 10.1 mg/dL for T-carriers vs. non-T-carriers in combined groups, p 5 0.045). In multivariate analysis adjusted for age, gender, BMI, diabetes and smoking status the presence of the Tth111I-T allele remained significantly associated with a lower CAD risk, but this significance was lost when the analysis was additionally adjusted for the logarithm of HDL-c concentration (Table 3). No significant associations between CAD and N363S or ER22/23EK polymorphisms were found. No significant univariate associations were found among Tth111I, N363S or ER22/23EK genotypes, alleles or their haplotypes and the severity of CAD when subgroups with SVD and MVD were compared ( p O0.05, data not shown); however, the variant homozygous TT genotype of Tth111I proved to be less common on the borderline of significance in MVD than SVD (5.9 vs. 14.1%, p 5 0.075). A negative association between the Tth111I T-T genotype and MVD was significant ( p 5 0.036) in multivariate analyses adjusted for age, gender, BMI and smoking status (Table 4), but additional adjustment of the multivariate model for diabetes caused loss of significance of the association (Table 4). Association among several metabolic profile parameters and the Tth111I, N363S and ER22/23EK variants was assessed. Allele 363S was significantly associated with DM (24.4 vs. 10.9%, carriers in DM and non-DM subjects, respectively, p 5 0.027), whereas the TT genotype of Tth111I was not found among subjects with DM and was present only in non-DM subjects (0 vs. 10.9%, p 5 0.012). Associations of DM with two NR3C1 loci can be explained by linkage disequilibrium because the TT genotype of Tth111I was never accompanied by the G allele of N636S.
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Gora˛cy et al./ Archives of Medical Research 44 (2013) 27e33
Table 2. Tth111I, N363S, ER22/23EK genotypes in patients with and without CAD Control group (n 5 113) Polymorphism Tth111I genotype CC CT TT Tth111I allele C T N363S genotype AA GA GG N363S allele A G ER22/23EK genotype GG GA AA ER22/23EK allele G A Tth111I /ER22/23EK/N363S diplotype CGA/CGA CGA/TGA TGA/TGA CGA/CGG TGA/CGG CGA/TAA TGA/TAA Tth111I /ER22/23EK/N363S haplotype CGA TGA CGGc TAAd
CAD group (n 5 197)
pa
Compared genotypes, or alleles
OR (95% CI)
pb
TTþCT vs. CC TT vs. CTþCC TT vs. CC
0.542 (0.337e0.873) 0.933 (0.424e2.052) 0.661 (0.287e1.524)
0.013 0.842 0.376
T vs. C
0.705 (0.499e0.997)
0.050
GA vs. AA
1.075 (0.536e2.155)
1.0
G vs. A
1.070 (0.545e2.094)
1.0
GA vs. GG
0.567 (0.113e2.858)
0.67
A vs. G
0.570 (0.114e2.850)
0.67
n
%
n
%
40 62 11
35.40 54.87 9.73
99 80 18
50.25 40.61 9.14
142 84
62.83 37.17
278 116
70.56 29.44
99 14 0
87.61 12.39 0.0
171 26 0
86.80 13.20 0.0
212 14
93.81 6.19
368 26
93.40 6.60
110 3 0
97.3% 2.7% 0%
194 3 0
98.5% 1.5% 0%
223 3
98.7% 1.3%
391 3
99.2% 0.8%
35 51 10 5 9 2 1
31.0% 45.1% 8.9% 4.4% 8.0% 1.8% 0.9%
84 68 16 15 11 1 2
42.6% 34.5% 8.1% 7.6% 5.6% 0.5% 1.0%
0.26
CGA carriers vs. others TGA carriers vs. others CGG carriers vs. others TAA carriers vs. others
1.246 0.574 1.075 0.567
(0.668e2.324) (0.358e0.921) (0.536e2.155) (0.113e2.858)
0.52 0.024 1.0 0.67
128 81 14 3
56.6% 35.8% 6.2% 1.3%
252 113 26 3
64.0% 28.7% 6.6% 0.8%
0.25
CGA vs. other TGA vs. other CGG vs. other TAA vs. other
1.359 0.720 1.070 0.570
(0.973e1.897) (0.508e1.020) (0.545e2.094) (0.114e2.850)
0.073 0.072 1.0 0.67
0.034
-
-
c test. Fisher exact. c Equivalent to A1220G allele G. d Equivalent to G198A allele A. a 2 b
Therefore, the association was also observed when carriers of two copies of TGA haplotype (TGA/TGA diplotype) were compared with other subjects (0 vs. 9.8% TGA/TGA
carriers in DM and non-DM subjects, respectively, p 5 0.020). The presence of the 363S allele was significantly associated with a higher prevalence of DM in the univariate
Table 3. Uni- and multivariate logistic regression analysis of association between the presence of Tth111I T allele and CAD
Table 4. Uni- and multivariate logistic regression analysis of association between the presence of Tth111I TT genotype and MVD in CAD patients
Model
OR
95% CI
p
Univariate Multivariatea Multivariateb
0.54 0.59 0.64
0.34e0.88 0.35e0.98 0.38e1.08
0.012 0.041 0.093
a
Adjusted for age, gender, BMI, diabetes and smoking status. Adjusted for age, gender, BMI, diabetes, smoking status and HDLcholesterol. b
Model
OR
95% CI
p
Univariate Multivariatea Multivariateb
0.38 0.33 0.38
0.14e1.04 0.12e0.94 0.13e1.08
0.057 0.036 0.068
a
Adjusted for age, gender, BMI and smoking status. Adjusted for age, gender, BMI, smoking status and diabetes.
b
Glucocorticoid Receptor Genes and Coronary Artery Disease
(OR 2.63, 95% CI: 1.20e5.77; p 5 0.015) and multivariate logistic regression model adjusted for age, gender, BMI, smoking status and CAD status (OR 3.33, 95% CI: 1.44e7.69; p 5 0.005). No association between metabolic profile and the ER22/ 23EK polymorphism was found (data not shown), but it should be noted that only six heterozygotes were detected. We conducted a separate analysis to compare hypertensive and normotensive patients. No associations were found between Tth111I, N363S or ER22/23EK-NR3C1 genotypes, alleles or their haplotypes and HT ( p O0.2, data not shown). Discussion This study evaluates the role of NR3C1 gene Tth111I, ER22/ ER23 and N363S polymorphisms and their association with CAD and risk factors in the Polish population. The analysis has revealed a protective role for the Tth111I-NR3C1 gene polymorphism against CAD. Carriers of the Tth111I allele T are significantly less common in the CAD group, but note a lack of association between ER22/ER23, N363S and CAD in our study. However, analysis of haplotypes encompassing all three loci revealed a significant protective role for the TGA haplotype. Prevalence of the NR3C1 gene polymorphisms in SVD or MVD was also investigated. It is known that age or diabetes is an important risk factor for the development of MVD (20). In our study an association was found between homozygous Tth111I and MVD and, to the best of our knowledge, this result has not been previously demonstrated. Therefore, the effects of the NR3C1 gene on MVD remains to be elucidated and replication of these studies in different population are needed. Literature reports have sometimes failed to demonstrate an effect of Tth111I polymorphism on the development of CAD. For example, van Rossum et al. (10) noted that the Tth111I polymorphism was not associated with altered GC sensitivity, but association with GC resistance and beneficial metabolic profile (low insulin and TC level) were observed in carriers of the ER22/23K and Tth111I polymorphisms. These findings have yet to be clarified. Previous studies have reported associations between NR3C1 gene polymorphisms and CAD, and these have been linked to altered metabolic profile. Lin at al. (5) showed that CAD is associated with the 363SeNR3C1 allele in a white Anglo-Celtic population. In a large Dutch population the common NR3C1 gene haplotype 3 (GGACG; ER22/23EK/ N363S/BcII/GR-9b) was found to be associated with an increased risk of MI and CHD. Additionally, homozygous carriers of haplotype 3 showed significantly higher levels of CRP and IL-6 (21). Van Rossum et al. (22) showed that polymorphisms of the NR3C1 gene may be involved in the modulation of the inflammatory process by such factors as CRP. In this study, ER22/EK23 carriers had lower CRP levels, and the authors suggested that these findings may possibly reflect a beneficial cardiovascular status (22).
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In contrast, in The Leiden 85-plus Study the authors show that in elderly subjects the ER22/23K and N363S polymorphisms, but not the BcII SNP in the NR3C1 gene, have an influence on metabolic profile (including a trend for high CRP concentrations). However, the noticed differences in metabolic profile do not affect the prevalence of cardiovascular pathologies and cardiovascular disease mortality during older ages (17). In our previous study, no association was found between polymorphisms of proinflammatory cytokines such as IL1B C(-31)T/IL1RN (VNTR) or their haplotypes and CAD in the Polish population, but an association with hypertension was noticed (23). It is known that inflammatory processes play a key role in development of atherosclerosis, and glucocorticoids are important regulators of the inflammatory process and may contribute to atherogenesis (24,25). Previously published results in other populations suggest that ER22/23EK carriers are relatively more resistant to cortisol than noncarriers, resulting in a better metabolic health profile (6). However, Koeijovoets et al. (13), in a high-risk population for cardiovascular disease with familial hypercholesterolemia (FH), observed no significant association between ER22/23EK polymorphism and CAD, and the effect of this variant on CAD risk was significantly different between males and females. In the current study we noted that the T allele of the Tth111I polymorphism of the NR3C1 gene occurred more frequently in females. It should also be noted that female carriers of the T allele of Tth111I had higher HDL-c levels. The possible mechanism of this finding may be explained by variations in circulating sex steroid concentrations. Animal experimental studies indicate that estrogen primarily exerts stimulatory effects on stress-induced ACTH and glucocorticoid release (26). The strength of our study is determined by the well-documented homogeneous cohort of individuals undergoing angiography in patients balanced with the control group. However, there is a limitation in our study of no information on postmenopausal status. This result may suggest that carriers of the Tth111I-Tallele, in combination with other factors, may partly protect from vascular damage in our population. It is also known that the general frequency of polymorphisms varies greatly among ethnic populations; therefore, results vary from population to population (10,27). The importance of known risk factors that are involved in development of CAD should be further emphasized. Furthermore, researchers reported correlations between GR gene polymorphisms and risk factors for coronary disease in different populations, especially with those overweight or obese (7,28,29). However, other studies demonstrated no association between NR3C1 gene polymorphisms and risk factors such as HT, diabetes or plasma insulin and TC levels (5,15). Dobson et al. (14) showed no association of the 363S allele with BMI, plasma lipid levels or glucose tolerance status in the Caucasian population. In accordance with this observation, in our study associations among cardiovascular risk factors (such as BMI, plasma
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Gora˛cy et al./ Archives of Medical Research 44 (2013) 27e33
lipid levels or hypertension) and Tth111I, N363S or ER22/ 23EK NR3C1 gene polymorphisms were not found. In the Polish population, risk factors such as diet, smoking habits, and unfavorable lifestyle are particularly strongly expressed and may overshadow the impact of genetic factors. However, in our population a significant association between the 363S allele and DM was noted. The hyperglycemia of type 2 diabetes is caused by improper function of the pancreas, increased hepatic glucose production and peripheral insulin resistance (28,30). These metabolic processes can be modulated by glucocorticoids (31,32). The N363S polymorphism shows an increased transactivating capacity in vitro and is associated with increased sensitivity to glicocorticosteroids in vivo (4,33). The exact mechanism has not been clarified yet, but Manenschijn et al. (34) suggest that the N363S phenotype may be the result of a differential effect on gene regulation. Additionally, van Rossum (10) indicated that a functional N363S polymorphism could be classified as having glucocorticoid hypersensitive effects, and such a mechanism in our population may play a significant role. CAD is a complex disease and genetic components related to its development are based on small to moderate effects of many genes. Genetic factors may have a weaker potency than the environment, and their impact is a result of a combined operation of genetic and environmental, social, economic, cultural factors that have an influence on the development of CAD. In conclusion, the present study suggests that the Tth111I polymorphism may play a protective role on the development of CAD and that homozygous carriers of the Tth111I variant could be protected from the development of MVD. Additionally, the N363S polymorphisms may influence the development of diabetes in the Polish population. References 1. Bjerntrop P, Holm G, Rosmond R. Hypothalamic arousal, insulin resistance and type 2 diabetes mellitus. Diabet Med 1999;16:373e383. 2. Walker BR. Glucocorticoids and cardiovascular disease. Eur J Endocrinol 2007;157:545e559. 3. Ross R. Atherosclerosis—an inflammatory disease. N Engl J Med 1999;34:115e126. 4. Huizenga NA, Koper JW, de Lange P, et al. A polymorphism in the glucocorticoid receptor gene may be associated with and increased sensitivity to glucocorticoids in vivo. J Endocrinol Metab 1998;83:144e151. 5. Lin RC, Wang WY, Morris BJ. Association of coronary artery disease with glucocorticoid receptor N363S variant. hypertension 2003;41:404e407. 6. van Rossum EF, Koper JW, Van Den Beld AW, et al. A polymorphism in the glucocorticoid receptor gene, which decreases sensitivity to glucocorticoids in vivo, is associated with low insulin and cholesterol levels. Diabetes 2002;52:3128e3134. 7. Di Blasio AM, van Rossum EF, Maestrini S, et al. The relation between two polymorphism in the glucocorticoid receptor gene and body mass index, blood pressure and cholesterol in obese patients. Clin Endocrinol (Oxf) 2003;59:68e74. 8. Malchoff DM, Brufsky A, Reardon G, et al. A mutation of the glucocorticoid receptor in primary cortisol resistance. J Clin Invest 1993;91: 1918e1925.
9. Karl M, Lamberts SW, Detera Wadleigh SD, et al. Familial glucocorticoid resistance caused by a splice site deletion in the human glucocorticoid receptor gene. J Clin Endocrinol Metab 1993;76:683e689. 10. van Rossum EF, Lamberts SW. Polymorphisms in the glucocorticoid receptor gene and their association with metabolic parameters and body composition. Recent Prog Horm Res 2004;59:333e357. 11. W€ust S, Van Rossum EF, Federenko IS, et al. Common polymorphisms in the glucocorticoid receptor gene are associated with adrenocortical responses to psychosocial stress. J Clin Endocrinol Metab 2004;59:565e573. 12. Derijk RH, de Kloet ER. Corticosteroid receptor polymorphism: determinants of vulnerability and resilience. Eur J Pharmacol 2008;583: 303e311. 13. Koeijvoets KCM, van der Net JB, van Rossum EFC, et al. Two common haplotypes of the of the glucocorticoid receptor gene are associated with increased susceptibility to cardiovascular disease in men with familial hypercholesterolemia. J Clin Endocrinol Metab 2008;93:4902e4908. 14. Dobson MG, Redfern CP, Unwin N, et al. The N363S polymorphism of the glucocorticoid receptor: potential contribution to central obesity in men and lack of association with other risk factors for coronary heart disease and diabetes mellitus. J Clin Endocrinol Metab 2001; 86:2270e2274. 15. Rosmond R, Bouchard C, Biorntorp P. Tsp509I polymorphism in exon 2 of the glucocorticoid receptor gene in relation to obesity and cortisol secretion: cohort study. BMJ 2001;322:652e653. 16. Echwald SM, Sorensen TI, Andersen T, et al. The Asn363Ser variant of the glucocorticoid receptor gene is not associated with obesity or weight gain in Danish men. Int J Obes Relat Met Disord 2001;25: 1563e1565. 17. Kunings M, Mooijaart SP, Slagboom PE, et al. Genetic variants in the glucocorticoid receptor gene (NR3C1) and cardiovascular disease risk. The Leiden 85-plus Study. Biogerontology 2006;7:231e238. 18. Otte Ch, W€ust S, Zhao S, et al. Glucocorticoid receptor gene, lowgrade inflammation, and heart failure: the Heart and Soul Study. J Clin Endocrinol Metab 2010;95:2885e2891. 19. Guo SW, Thompson EA. Performing the exact test of Hardy-Weinberg proportion for multiple alleles. Biometrics 1992;48:361e372. 20. Milicevic Z, Raz I, Beattie SD, et al. Natural history of cardiovascular disease In patients with diabetes: role of hyperglycemia. Diabetes Care 2008;31:155e160. 21. Van den Akker ELT, Koper JW, van Rossum EFC, et al. Glucocorticoid receptor and risk of cardiovascular disease. Arch Intern Med 2008;168:33e39. 22. Van Rossum EF, Feelders RA, van den Beld AW, et al. Association of the ER22/23EK polymorphism in the glucocorticoid receptor gene with survival and C-reactive protein levels in elderly men. Am J Med 2004;117:158e162. 23. Gora˛cy J, Gora˛cy I, Safranow K, et al. Lack of association of inteleukin1 gene cluster polymorphisms with angiographically documented coronary artery disease: demonstration of association with hypertension in the Polish population. Arch Med Res 2011;42:426e432. 24. Libby P. Inflammation in atherosclerosis. Nature 2002;420:868e874. 25. Rhen T, Cidlowski JA. Antiinflammatory action of glucocorticoidsnew mechanism for old drugs. N Engl J Med 2005;353:1711e1723. 26. Handa RJ, Burgess LH, Kerr JE, et al. Gonadal steroid hormone receptors and sex differences in the hypothalamo-pituitary-adrenal axis. Horm Behav 1994;28:464e476. 27. Syed AA, Irving JA, Redfern CP, et al. Low prevalence of the N363S polymorphism of the glucocorticoid receptor in South Asians living in the United Kingdom. J Clin Endocrinol Metab 2004;89:232e235. 28. Roussel R, Reis AF, Dubois-Laforgue D, et al. The N363S polymorphism in the glucocorticoid receptor gene is associated with overweight in subject with type 2 diabetes mellitus. Clin Endocrinol 2003;59:237e241.
Glucocorticoid Receptor Genes and Coronary Artery Disease 29. Lin RC, Wang WY, Morris BJ. High penetrance, overweight and glucocorticoid receptor variant: case-control study. Br Med J 1999; 319:1337e1338. 30. DeFronzo RA. Pathogenesis of type 2 diabetes: metabolic and molecular implication for identifying diabetes genes. Diabetes Rev 1997;5:177e269. 31. Lambillotte C, Gilon P, Henquin JC. Direct glucocorticoid inhibition of insulin secretion. An in vitro study of dexamethasone effects in mouse islets. J Clin Invest 1997;99:414e423.
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32. Andrews RC, Walker BR. Glucocorticoids and insulin resistance: old hormones, new targets. Clin Sci 1999;96:513e523. 33. Koper JW, Stolk RP, de Lange P, et al. Lack of associations between five polymorphisms in the human glucocorticoid receptor gene and glucocorticoid resistance. Hum Genet 1997;99:663e668. 34. Manenschijn L, van der Akker ELT, Lamberts SWJ, et al. Clinical features associates with glucocorticoid receptor polymorphisms. Ann NY Acad Sci 2009;1179:179e198.
ORIGINAL ARTICLE
Association of Glucocorticoid Receptor Gene NR3C1 Genetic Variants with Angiographically Documented Coronary Artery Disease and Its Risk Factors a _ Iwona Gora˛cy,a Jaros1aw Gora˛cy,b Krzysztof Safranow,c Karolina Skonieczna-Zydecka, and Andrzej Ciechanowicza a
Department of Clinical and Molecular Biochemistry, Pomeranian Medical University, Szczecin, Poland b Clinic of Cardiology, Pomeranian Medical University, Szczecin, Poland c Department of Biochemistry and Medical Chemistry, Pomeranian Medical University, Szczecin, Poland Received for publication June 21, 2012; accepted October 30, 2012 (ARCMED-D-12-00315).
Background and Aims. Glucocorticoids and their receptors are involved in inflammation and many cardiovascular risk factors. We examined associations of Tth111I, N363S and ER22/23EK NR3C1 gene polymorphisms and haplotypes, with coronary artery disease (CAD), severity of CAD (single-vessel vs. multivessel disease) and risk factors. Methods. Three hundred ten individuals were submitted to coronary angiography. Selected genotypes were determined by PCR-RFLP. Results. Carriers of the Tth111I allele T were found significantly less often in the CAD compared with the non-CAD group (49.7 vs. 64.6%, p 5 0.013); this association was similar for TGA haplotype carriers (49.2 vs. 62.8%, p 5 0.024); the T allele was more frequent in females (66.3 vs. 51.1%, p 5 0.020) and its presence was associated with higher levels of HDL-cholesterol (46.6 12.7 vs. 43.5 10.1 mg/dL for T-carriers vs. noncarriers, p 5 0.045). The TT genotype proved to be less common in MVD than SVD (5.9 vs. 14.1%, p 5 0.075). The 363S allele was significantly associated with diabetes mellitus (DM) (24.4 vs. 10.9%; carriers in DM and non-DM subjects, respectively, p 5 0.027), the TT genotype or TGA/TGA diplotype (in which the 363S allele was absent) were less frequent in DM than in non-DM subjects ( p 5 0.012 for Tth111ITT and p 5 0.020 for TGA/TGA diplotype). No significant associations between CAD and N363S or ER22/23EK polymorphisms were found. Conclusions. Our results suggest that the Tth111I NR3C1 polymorphism may play a protective role in the development of CAD, and homozygous TT in development of MVD. The N363S polymorphisms may contribute to the development of diabetes in the Polish population. Ó 2013 IMSS. Published by Elsevier Inc. Key Words: Glucocorticoid receptor, Coronary artery disease, Risk factors, Polymorphism.
Introduction Glucocorticoids (GC) are regulators of the immune system, inflammatory processes, and many other process involved in fat and glucose metabolism (1,2). Inflammatory processes have been recognized to play a key role in the pathogenesis of
Address reprint requests to: Iwona Gora˛cy, Department of Clinical and Molecular Biochemistry, Pomeranian Medical, University, Szczecin, Al. Powsta nc ow Wlkp. 72, 70-111 Szczecin, Poland; Phone: þ48 91 466 14 90; FAX: þ48 91 466 14 92; E-mail: [email protected]
atherosclerosis and coronary artery disease (CAD) (3). It is known that sensitivity to glucocorticoids varies considerably between individuals (4) and several studies have showed that high levels of glucocorticoids result in unfavorable cardiovascular risk factors such as central obesity, steroidinduced diabetes, or dyslipidemia (5e7). The glucocorticoid receptor (GR) is a crucial factor in glucocorticoid responses. GR gene mutations lead to reduced intracellular concentrations or activity of GR in glucocorticoid target tissues (8,9). One previous report described that polymorphisms in the glucocorticoid
0188-4409/$ - see front matter. Copyright Ó 2013 IMSS. Published by Elsevier Inc. http://dx.doi.org/10.1016/j.arcmed.2012.10.020
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receptor, NR3C1 gene: ER22/23EK; BcII C/G, N363S; and 9b A/G, modulate glucocorticoid sensitivity (10e12) and two polymorphisms in the NR3C1 gene, the N363S and BcII variants, have been associated with an increased sensitivity to glucocorticoids in vivo; carriers had lower cortisol levels after a dexamethasone suppression test (13,4). Genetic variation in the NR3C1 gene has the potential to predispose to several risk factors for atherosclerosis: higher body mass index (BMI), elevated plasma lipid levels, lower C-reactive protein (CRP) levels, and insulin resistance (4,6). In addition genetic variants in the NR3C1 gene have shown an association with CAD and essential hypertension (HT) (5,7). However, there are disparities in the published literature. In some studies no association was observed (14e16). For example, three polymorphisms (ER22/ 23ER, N363S, and BclI) were studied in a population of elderly persons together with the risk for cardiovascular disease and no association was found (17). In another study of patients with stable CAD and haplotype analysis of the NR3C1 gene, an association was found with self-reported heart failure or systolic dysfunction (18). In a further multicenter study, two common GR gene haplotypes modified cardiovascular disease susceptibility in males (13). Considering these important but contrasting findings, additional studies on the relationship between NR3C1 gene polymorphisms and CAD or its risk factors (diabetes, HT, dyslipidemia) are needed. Therefore, in the present study we assessed the impact of the Tth111I, N363S, ER22/ 23EK polymorphisms of the NR3C1 gene and reconstructed their haplotypes on the prevalence of CAD or its risk factors in a sample of Polish patients subjected to coronary angiography, Associations between the polymorphisms and the extent of coronary vessel occlusion were also studied.
Materials and Methods Study Group The study protocol was approved by the local ethics committee with formal informed consent signed by all the participants. The patients were randomly recruited from the in-patient Cardiology Department, Pomeranian Medical University, Szczecin, Poland, with 310 individuals presenting with a history of CAD or stenocardiac symptoms screened for this study. Of these, 197 patients with $50% occlusion of at least one major coronary artery lumen in angiography comprised the CAD group, regardless of further possible treatment. The group was divided into 78 patients with single-vessel disease (SVD) and 119 with multivessel disease (MVD). The control group (non-CAD) consisted of the remaining 113 subjects without changes in coronary arteries. Patients with a history of myocardial infarction or with clinical diagnosis of cardiomyopathy, coagulopathy, collagenosis and chronic inflammatory disease were
excluded from the study. Arterial hypertension was defined as either systolic blood pressure O140 mmHg or diastolic blood pressure O90 mmHg or antihypertensive treatment; and diabetes mellitus as patients using antidiabetic medication or fasting plasma glucose O6.9 mmol/L. BMI was calculated as mass/height2. Patients were classified as current smokers if they reported a daily rate of more than five cigarettes. Otherwise, patients were classified as nonsmokers. Serum concentrations of total cholesterol (TC), triacylglycerols (TG), high-density lipoprotein cholesterol (HDL-c) and low-density lipoprotein cholesterol (LDL-c) were measured using enzymatic methods (Roche Diagnostics, Warsaw, Poland). Coronary angiography was performed according to standard procedures using Philips INTEGRIS HM 3000 and Philips ALURA (Philips, The Netherlands) devices. Genotyping Genomic DNA was extracted from peripheral blood leukocytes using the QIAamp DNA Mini Kit (QIAGEN, Hilden, Germany). For analysis of the Tth111I [C(-3807)T, rs 10052957)] polymorphism in the NR3C1 gene a polymerase chain reactionerestriction fragment length polymorphism (PCR/RFLP) method was designed with the following primer pair—forward: 5’-TATTTgTTgggTgCCTgCTATgTA-3’ and reverse 5’-gATgAACTCCAgTgTgCCAgAAAg3’ (TIB MOL BIOL, Poznan, Poland). The Tth111I NR3C1amplicons were subsequently digested using the PsyI restriction enzyme (MBI Fermentas, Vilnius, Lithuania). The PCR Tth111I NR3C1 gene product of 367 base pairs (bp) was cut into 278-bp and 89-bp fragments in the presence of C allele and 367 bp remained undigested in case of the T allele. DNA fragments that contained the N363S (A1220G, rs6195) NR3C1 polymorphism were amplified by PCR using primers: forward 5’-ATTCCCgTTggTTCCgAC-3’ and reverse 5’-ATCCCAggTCATTTCCCATC-3’ (TIB MOL BIOL). PCR-RFLP with the MunI restriction enzyme (MBI Fermentas) was performed. In the case of the G allele the final product of 125 bp remained undigested, whereas the A variant gave restriction fragments of 107 bp and 18 bp. For analysis of the ER22/ER23 (G198A/G200A, rs6190) polymorphism of NR3C1, PCR used forward 5’-gATTCggTCATTTCCCATC3’ and reverse 5’-ATCCCAggTCATTTCCCATC-3’ primers. Amplicons were subsequently digested using the MnlI restriction enzyme (MBI Fermentas). The PCR product (442 bp) was cut into fragments of 164 bp, 143 bp for the G200 allele and 178 bp, 164 bp for the A200 allele. Restriction fragments in each case were electrophoretically separated and visualized in ethidium bromide-stained 3% agarose gels. Statistical Analysis Quantitative variables were compared between groups using Kruskal-Wallis or Mann-Whitney tests. c2 or Fisher
Glucocorticoid Receptor Genes and Coronary Artery Disease
29
Table 1. Characteristics of patients with CAD, SVD, advanced disease (MVD) and without CAD (non-CAD) Parameter Male gender Age years BMI (kg/m2) Triacylglycerols (mg/dL) Total cholesterol (mg/dL) HDL-cholesterol (mg/dL) LDL-cholesterol (mg/dL) Smoking Diabetes Hypertension
Non-CAD (n 5 113)
CAD (n 5 197)
p CAD vs. non-CADa
SVD (n 5 78)
MVD (n 5 119)
p MVD vs. SVDa
59.0% 55.2 9.6 26.6 4.1 161.6 88.0 219.6 37.7 49.2 14.0 131.7 29.3 23.0% 8.9% 48.7%
81.7% 57.2 9.1 27.8 3.7 177 70.0 225.9 41.8 43.0 9.4 138.7 33.4 37.6% 17.8% 61.4%
!0.00002 0.082 0.017 0.0068 0.11 0.000055 0.0364 0.008 0.043 0.032
81.0% 54.9 8.0 27.8 3.7 181.8 96.2 222.5 40.6 43.9 9.9 134.9 31.3 36.7% 11.4% 54.4%
82.8% 58.7 9.5 27.8 3.7 174.0 67.3 228.3 42.5 42.4 8.9 141.2 34.6 37.7% 22.1% 65.6%
0.87 0.010 0.80 0.57 0.25 0.26 0.11 0.87 0.014 0.11
CAD, coronary artery disease; SVD, single-vessel disease; MVD, multivessel disease. Data are given as mean SD or %. a p values were calculated with Mann-Whitney test for quantitative variables and with Fisher exact test for qualitative variables.
exact test was used for qualitative variables (genetic data, gender, presence of diabetes, CAD or MVD and smoking status). An exact test was applied to assess conformity of the genotype distribution to Hardy-Weinberg law (19). A multivariate logistic regression model was used to analyze genotypeephenotype association after adjustment for potential confounders (age, gender, BMI, smoking status, presence of diabetes or CAD and HDL-c concentration). Haplotypes were analyzed using the Haploview 4.2 program; p !0.05 was considered statistically significant. Results Baseline characteristics of CAD and non-CAD subjects are shown in Table 1. Differences between patients with SVD and MVD are also presented in this Table 1. Frequency of male gender and value of BMI proved to be significantly higher in the CAD group when compared to controls ( p !0.00002 and p 5 0.017 respectively). Additionally, smoking, diabetes and HT were more common among CAD patients than controls ( p 5 0.008, p 5 0.043 and p 5 0.032, respectively), and serum concentrations of TG and LDL-c were higher among CAD patients than controls ( p 5 0.036 and p 5 0.0068, respectively); HDL-c was lower in CAD patients ( p 5 0.000055). A significant difference in the clinical and biochemical characteristics between patients with SVD and MVD was noted only for age and diabetes (Table 1). The Tth111I, N363S or ER22/23EKeNR3C1 genotypes (Table 2) were found to be in Hardy-Weinberg equilibrium in both CAD and non-CAD groups ( p O0.1). The three loci were then examined for the level of LD in the entire group (n 5 310). The estimated values of D’ were equal to 1 for each pair of loci. Frequencies of haplotypes (Tth111I-ER22/23EK-N363S) were 61.2% for CGA, 31.3% for TGA, 6.5% for CGG and 1.0% for TAA. We observed a significant association between CAD and genotypes of Tth111I (Table 2). Analysis revealed that carriers of the Tth111I allele T (CTþTT genotypes) were significantly less common in the CAD group than in
the non-CAD group (49.7 vs. 64.6%, p 5 0.013). This association was similar for carriers of the TGA haplotype that contain the Tth111I-T allele (49.2 vs. 62.8%, p 5 0.024). However, we also noted that the Tth111I-T allele occurred more frequently in females than in males (66.3 vs. 51.1%; T carriers for combined groups, p 5 0.020) and its presence was associated with a higher level of HDL-c (46.6 12.7 vs. 43.5 10.1 mg/dL for T-carriers vs. non-T-carriers in combined groups, p 5 0.045). In multivariate analysis adjusted for age, gender, BMI, diabetes and smoking status the presence of the Tth111I-T allele remained significantly associated with a lower CAD risk, but this significance was lost when the analysis was additionally adjusted for the logarithm of HDL-c concentration (Table 3). No significant associations between CAD and N363S or ER22/23EK polymorphisms were found. No significant univariate associations were found among Tth111I, N363S or ER22/23EK genotypes, alleles or their haplotypes and the severity of CAD when subgroups with SVD and MVD were compared ( p O0.05, data not shown); however, the variant homozygous TT genotype of Tth111I proved to be less common on the borderline of significance in MVD than SVD (5.9 vs. 14.1%, p 5 0.075). A negative association between the Tth111I T-T genotype and MVD was significant ( p 5 0.036) in multivariate analyses adjusted for age, gender, BMI and smoking status (Table 4), but additional adjustment of the multivariate model for diabetes caused loss of significance of the association (Table 4). Association among several metabolic profile parameters and the Tth111I, N363S and ER22/23EK variants was assessed. Allele 363S was significantly associated with DM (24.4 vs. 10.9%, carriers in DM and non-DM subjects, respectively, p 5 0.027), whereas the TT genotype of Tth111I was not found among subjects with DM and was present only in non-DM subjects (0 vs. 10.9%, p 5 0.012). Associations of DM with two NR3C1 loci can be explained by linkage disequilibrium because the TT genotype of Tth111I was never accompanied by the G allele of N636S.
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Table 2. Tth111I, N363S, ER22/23EK genotypes in patients with and without CAD Control group (n 5 113) Polymorphism Tth111I genotype CC CT TT Tth111I allele C T N363S genotype AA GA GG N363S allele A G ER22/23EK genotype GG GA AA ER22/23EK allele G A Tth111I /ER22/23EK/N363S diplotype CGA/CGA CGA/TGA TGA/TGA CGA/CGG TGA/CGG CGA/TAA TGA/TAA Tth111I /ER22/23EK/N363S haplotype CGA TGA CGGc TAAd
CAD group (n 5 197)
pa
Compared genotypes, or alleles
OR (95% CI)
pb
TTþCT vs. CC TT vs. CTþCC TT vs. CC
0.542 (0.337e0.873) 0.933 (0.424e2.052) 0.661 (0.287e1.524)
0.013 0.842 0.376
T vs. C
0.705 (0.499e0.997)
0.050
GA vs. AA
1.075 (0.536e2.155)
1.0
G vs. A
1.070 (0.545e2.094)
1.0
GA vs. GG
0.567 (0.113e2.858)
0.67
A vs. G
0.570 (0.114e2.850)
0.67
n
%
n
%
40 62 11
35.40 54.87 9.73
99 80 18
50.25 40.61 9.14
142 84
62.83 37.17
278 116
70.56 29.44
99 14 0
87.61 12.39 0.0
171 26 0
86.80 13.20 0.0
212 14
93.81 6.19
368 26
93.40 6.60
110 3 0
97.3% 2.7% 0%
194 3 0
98.5% 1.5% 0%
223 3
98.7% 1.3%
391 3
99.2% 0.8%
35 51 10 5 9 2 1
31.0% 45.1% 8.9% 4.4% 8.0% 1.8% 0.9%
84 68 16 15 11 1 2
42.6% 34.5% 8.1% 7.6% 5.6% 0.5% 1.0%
0.26
CGA carriers vs. others TGA carriers vs. others CGG carriers vs. others TAA carriers vs. others
1.246 0.574 1.075 0.567
(0.668e2.324) (0.358e0.921) (0.536e2.155) (0.113e2.858)
0.52 0.024 1.0 0.67
128 81 14 3
56.6% 35.8% 6.2% 1.3%
252 113 26 3
64.0% 28.7% 6.6% 0.8%
0.25
CGA vs. other TGA vs. other CGG vs. other TAA vs. other
1.359 0.720 1.070 0.570
(0.973e1.897) (0.508e1.020) (0.545e2.094) (0.114e2.850)
0.073 0.072 1.0 0.67
0.034
-
-
c test. Fisher exact. c Equivalent to A1220G allele G. d Equivalent to G198A allele A. a 2 b
Therefore, the association was also observed when carriers of two copies of TGA haplotype (TGA/TGA diplotype) were compared with other subjects (0 vs. 9.8% TGA/TGA
carriers in DM and non-DM subjects, respectively, p 5 0.020). The presence of the 363S allele was significantly associated with a higher prevalence of DM in the univariate
Table 3. Uni- and multivariate logistic regression analysis of association between the presence of Tth111I T allele and CAD
Table 4. Uni- and multivariate logistic regression analysis of association between the presence of Tth111I TT genotype and MVD in CAD patients
Model
OR
95% CI
p
Univariate Multivariatea Multivariateb
0.54 0.59 0.64
0.34e0.88 0.35e0.98 0.38e1.08
0.012 0.041 0.093
a
Adjusted for age, gender, BMI, diabetes and smoking status. Adjusted for age, gender, BMI, diabetes, smoking status and HDLcholesterol. b
Model
OR
95% CI
p
Univariate Multivariatea Multivariateb
0.38 0.33 0.38
0.14e1.04 0.12e0.94 0.13e1.08
0.057 0.036 0.068
a
Adjusted for age, gender, BMI and smoking status. Adjusted for age, gender, BMI, smoking status and diabetes.
b
Glucocorticoid Receptor Genes and Coronary Artery Disease
(OR 2.63, 95% CI: 1.20e5.77; p 5 0.015) and multivariate logistic regression model adjusted for age, gender, BMI, smoking status and CAD status (OR 3.33, 95% CI: 1.44e7.69; p 5 0.005). No association between metabolic profile and the ER22/ 23EK polymorphism was found (data not shown), but it should be noted that only six heterozygotes were detected. We conducted a separate analysis to compare hypertensive and normotensive patients. No associations were found between Tth111I, N363S or ER22/23EK-NR3C1 genotypes, alleles or their haplotypes and HT ( p O0.2, data not shown). Discussion This study evaluates the role of NR3C1 gene Tth111I, ER22/ ER23 and N363S polymorphisms and their association with CAD and risk factors in the Polish population. The analysis has revealed a protective role for the Tth111I-NR3C1 gene polymorphism against CAD. Carriers of the Tth111I allele T are significantly less common in the CAD group, but note a lack of association between ER22/ER23, N363S and CAD in our study. However, analysis of haplotypes encompassing all three loci revealed a significant protective role for the TGA haplotype. Prevalence of the NR3C1 gene polymorphisms in SVD or MVD was also investigated. It is known that age or diabetes is an important risk factor for the development of MVD (20). In our study an association was found between homozygous Tth111I and MVD and, to the best of our knowledge, this result has not been previously demonstrated. Therefore, the effects of the NR3C1 gene on MVD remains to be elucidated and replication of these studies in different population are needed. Literature reports have sometimes failed to demonstrate an effect of Tth111I polymorphism on the development of CAD. For example, van Rossum et al. (10) noted that the Tth111I polymorphism was not associated with altered GC sensitivity, but association with GC resistance and beneficial metabolic profile (low insulin and TC level) were observed in carriers of the ER22/23K and Tth111I polymorphisms. These findings have yet to be clarified. Previous studies have reported associations between NR3C1 gene polymorphisms and CAD, and these have been linked to altered metabolic profile. Lin at al. (5) showed that CAD is associated with the 363SeNR3C1 allele in a white Anglo-Celtic population. In a large Dutch population the common NR3C1 gene haplotype 3 (GGACG; ER22/23EK/ N363S/BcII/GR-9b) was found to be associated with an increased risk of MI and CHD. Additionally, homozygous carriers of haplotype 3 showed significantly higher levels of CRP and IL-6 (21). Van Rossum et al. (22) showed that polymorphisms of the NR3C1 gene may be involved in the modulation of the inflammatory process by such factors as CRP. In this study, ER22/EK23 carriers had lower CRP levels, and the authors suggested that these findings may possibly reflect a beneficial cardiovascular status (22).
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In contrast, in The Leiden 85-plus Study the authors show that in elderly subjects the ER22/23K and N363S polymorphisms, but not the BcII SNP in the NR3C1 gene, have an influence on metabolic profile (including a trend for high CRP concentrations). However, the noticed differences in metabolic profile do not affect the prevalence of cardiovascular pathologies and cardiovascular disease mortality during older ages (17). In our previous study, no association was found between polymorphisms of proinflammatory cytokines such as IL1B C(-31)T/IL1RN (VNTR) or their haplotypes and CAD in the Polish population, but an association with hypertension was noticed (23). It is known that inflammatory processes play a key role in development of atherosclerosis, and glucocorticoids are important regulators of the inflammatory process and may contribute to atherogenesis (24,25). Previously published results in other populations suggest that ER22/23EK carriers are relatively more resistant to cortisol than noncarriers, resulting in a better metabolic health profile (6). However, Koeijovoets et al. (13), in a high-risk population for cardiovascular disease with familial hypercholesterolemia (FH), observed no significant association between ER22/23EK polymorphism and CAD, and the effect of this variant on CAD risk was significantly different between males and females. In the current study we noted that the T allele of the Tth111I polymorphism of the NR3C1 gene occurred more frequently in females. It should also be noted that female carriers of the T allele of Tth111I had higher HDL-c levels. The possible mechanism of this finding may be explained by variations in circulating sex steroid concentrations. Animal experimental studies indicate that estrogen primarily exerts stimulatory effects on stress-induced ACTH and glucocorticoid release (26). The strength of our study is determined by the well-documented homogeneous cohort of individuals undergoing angiography in patients balanced with the control group. However, there is a limitation in our study of no information on postmenopausal status. This result may suggest that carriers of the Tth111I-Tallele, in combination with other factors, may partly protect from vascular damage in our population. It is also known that the general frequency of polymorphisms varies greatly among ethnic populations; therefore, results vary from population to population (10,27). The importance of known risk factors that are involved in development of CAD should be further emphasized. Furthermore, researchers reported correlations between GR gene polymorphisms and risk factors for coronary disease in different populations, especially with those overweight or obese (7,28,29). However, other studies demonstrated no association between NR3C1 gene polymorphisms and risk factors such as HT, diabetes or plasma insulin and TC levels (5,15). Dobson et al. (14) showed no association of the 363S allele with BMI, plasma lipid levels or glucose tolerance status in the Caucasian population. In accordance with this observation, in our study associations among cardiovascular risk factors (such as BMI, plasma
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Gora˛cy et al./ Archives of Medical Research 44 (2013) 27e33
lipid levels or hypertension) and Tth111I, N363S or ER22/ 23EK NR3C1 gene polymorphisms were not found. In the Polish population, risk factors such as diet, smoking habits, and unfavorable lifestyle are particularly strongly expressed and may overshadow the impact of genetic factors. However, in our population a significant association between the 363S allele and DM was noted. The hyperglycemia of type 2 diabetes is caused by improper function of the pancreas, increased hepatic glucose production and peripheral insulin resistance (28,30). These metabolic processes can be modulated by glucocorticoids (31,32). The N363S polymorphism shows an increased transactivating capacity in vitro and is associated with increased sensitivity to glicocorticosteroids in vivo (4,33). The exact mechanism has not been clarified yet, but Manenschijn et al. (34) suggest that the N363S phenotype may be the result of a differential effect on gene regulation. Additionally, van Rossum (10) indicated that a functional N363S polymorphism could be classified as having glucocorticoid hypersensitive effects, and such a mechanism in our population may play a significant role. CAD is a complex disease and genetic components related to its development are based on small to moderate effects of many genes. Genetic factors may have a weaker potency than the environment, and their impact is a result of a combined operation of genetic and environmental, social, economic, cultural factors that have an influence on the development of CAD. In conclusion, the present study suggests that the Tth111I polymorphism may play a protective role on the development of CAD and that homozygous carriers of the Tth111I variant could be protected from the development of MVD. Additionally, the N363S polymorphisms may influence the development of diabetes in the Polish population. References 1. Bjerntrop P, Holm G, Rosmond R. Hypothalamic arousal, insulin resistance and type 2 diabetes mellitus. Diabet Med 1999;16:373e383. 2. Walker BR. Glucocorticoids and cardiovascular disease. Eur J Endocrinol 2007;157:545e559. 3. Ross R. Atherosclerosis—an inflammatory disease. N Engl J Med 1999;34:115e126. 4. Huizenga NA, Koper JW, de Lange P, et al. A polymorphism in the glucocorticoid receptor gene may be associated with and increased sensitivity to glucocorticoids in vivo. J Endocrinol Metab 1998;83:144e151. 5. Lin RC, Wang WY, Morris BJ. Association of coronary artery disease with glucocorticoid receptor N363S variant. hypertension 2003;41:404e407. 6. van Rossum EF, Koper JW, Van Den Beld AW, et al. A polymorphism in the glucocorticoid receptor gene, which decreases sensitivity to glucocorticoids in vivo, is associated with low insulin and cholesterol levels. Diabetes 2002;52:3128e3134. 7. Di Blasio AM, van Rossum EF, Maestrini S, et al. The relation between two polymorphism in the glucocorticoid receptor gene and body mass index, blood pressure and cholesterol in obese patients. Clin Endocrinol (Oxf) 2003;59:68e74. 8. Malchoff DM, Brufsky A, Reardon G, et al. A mutation of the glucocorticoid receptor in primary cortisol resistance. J Clin Invest 1993;91: 1918e1925.
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