Introduction:
Today the primary reasons of human mortality worldwide are noninfectious diseases among which the leading place is taken by the cardiovascular system diseases (CSD). For several decades CSD are the prevailing factor resulting in morbidity and mortality among the able-bodied population in industrially developed countries. In 2012 the CSD contribution to the overall mortality of the population was 1056 thousand people, that is equal 55.4% of the total number of deaths, in Russia. Furthermore, the mortality rate from these diseases in Russia significantly exceeds this index in Western Europe and North America countries.(1)
The coronary heart disease (CHD) is the most common abnormality among CSD, which occurs mainly due to atherosclerotic lesions of arteries. In multiple studies it was shown that CHD has both environmental and genetic etiology (2), nevertheless the concrete causes are still to be discovered.(3,4) Significant evidence demonstrate great contribution of genetic factors, at the same time received data about the various genes contribution to CHD development are controversial and require further studies in different populations.(5,6)
The genes coding for the proteins of the renin-angiotensin system (RAS) may assist in the development of CHD, due to their core role in the regulation of blood pressure and vascular remodeling.(7) Angiotensinogen is the integral component of RAS system. It is considered to be involved in the pathogenesis of hypertension and CHD.(8,9) Many studies have established and replicated the association of the two polymorphisms (M235T, T174M) in the AGT gene with CHD, and it was determined that their frequency differ in various populations. However, results from these reports are conflicting, and more studies are required to further confirm these findings.(8-11)
Thus the one of the key priorities of the present and future medicobiological studies are the disease causation understanding and enhancement of the opportunities in the field of the CHD prevention, diagnostics and treatment in different populations. Therefore we designed case-control study to examine the relationship between the genetic markers in the renin-angiotensin system and the presence of CHD in individuals residing in Rostov-on-Don, Russia.
Materials and Methods
The total population of this study consisted of 209 Russian individuals, residents of Rostov-on-Don, was divided into two groups: 50 angiographically verified CHD patients and 159 healthy subjects (Table 1). Patients with CHD were recruited from patients admitted to the cardiology section of Rostov regional clinical hospital (Rostov-on-Don, Russia). Documented stable CHD was diagnosed by the clinical evaluation and coronary angiography or occurrence of myocardial infarction (MI) as defined by WHO criteria. The control subjects were randomly selected and were age matched with CHD patients. They had no history of CHD, MI or stroke. Informed consent was obtained from all participants and the design of the study was approved by the local ethics committee.
Table 1: Clinical characteristics of study participants |
Clinical characteristics |
CHD (n=50) |
Control (n=159) |
Age (mean ± standard deviation) |
61.2 ± 2.9 |
55.5 ± 9.0 |
Male (men/women) |
35/15 |
79/80 |
Ever smoked (%) |
98 |
9 |
Type-2 diabetes (%) |
38 |
0 |
Systolic blood pressure (mm Hg, mean ± standard deviation) |
144.1 ± 1.82 |
137.1 ± 1.18 |
Diastolic blood pressure (mm Hg, mean ± standard deviation) |
88.8 ± 0.97 |
81.7 ± 0.59 |
| CHD = coronary heart disease. |
CHD risk factors were determined according to the criteria of the European society of cardiology: a hypertensive condition was diagnosed when systolic blood pressure values were = 139 mm Hg and/or diastolic blood pressure values = 89 mm Hg or when being medicated against hypertension; subjects were considered smokers when consuming more than five cigarettes per day or had stopped smoking at least 1 year before sample collection or non-smokers when never smoked.
Isolation of DNA and genetic polymorphism detection. DNA was extracted from leukocytes by standard procedures.(12) The identification of the AGT gene T174M and M235T polymorphisms was carried out by allele-specific PCR according to the protocol (Genetic Polymorphism Detection Kit by Allele-specific PCR in human genome «SNP-EXPRESS», Lytech Company, Moscow, Russia). The analysis is based on the amplification reaction with two pairs of the allele-specific primers. PCR products were separated on 3% agarose gel stained with ethidium bromide. Then the gel was visualized under UV transilluminator (GelDoc, BioRad).
Genotype frequencies in cases and controls were tested for Hardy–Weinberg equilibrium using Hardy-Weinberg equilibrium calculator on http://www.oege.org/software/hwe-mr-calc.shtml (13), and the deviation between the observed and expected frequencies was tested for significance using the Chi-square test (?2) using BIOSTAT. The odds ratios (ORs) and 95% confidence intervals (CIs) were calculated regarding the presence of CHD with respect to the existence of polymorphism. The difference was considered significant at P < 0.05.(14)
Results
The genotypes frequencies of the AGT gene were in agreement with Hardy–Weinberg equilibrium estimated by ?2 in both groups. The allele frequencies were q (235T) = 0.36 and 0.15, q (174?) = 0.12 and 0.16 for patients and controls, respectively. The CHD group showed significantly increased frequencies of the AGT MT and TT genotypes compared with controls (P = 0.03 and 0.001, respectively), whereas the analyses did not show a statistically significant difference for the AGT ?174? polymorphism between cases and controls (Table 2). Subjects with AGT gene M235T polymorphism MT and TT genotypes were significantly more likely to develop CHD (OR = 2.63, 95% CI = 1.1-6.1, P = 0.03 and OR = 4.88, 95% CI = 1.1-6.1, P = 0.001, respectively), than individuals with AGT gene ?174? polymorphism TM and MM genotypes (OR = 0.65 95% CI = 0.28-1.5, P > 0.05 and OR = 0.96 95% CI = 0.04-10.8, P > 0.05, respectively).
Table 2: AGT genotypes distribution in cases and controls |
Gene polymorphism |
Genotype |
CHD
n (%) |
Control
n (%) |
OR (CI 95%) |
P |
AGT-M235T |
MM |
25 (49.1) |
122 (76.5) |
- |
- |
MT |
14 (28.5) |
26 (16.2) |
2.63 (1.1-6.1) |
0.03 |
TT |
11 (22.4) |
11 (7.3) |
4.88 (1.1-6.1) |
0.001 |
AGT-T174M |
TT |
39 (77.6) |
112 (70.4) |
- |
- |
TM |
10 (20.4) |
44 (27.7) |
0.65 (0.28-1.5) |
> 0.05 |
MM |
1 (2.0) |
3 (1.9) |
0.96 (0.04-10.8) |
> 0.05 |
| CHD = coronary heart disease; AGT = angiotensinogen gene. |
Discussion
Among multiple AGT gene variants the most studied in relation to CHD are M235T and T174M. The genotype and allele frequencies of both AGT polymorphisms vary in different populations (Table 3). The 235T allele frequency lies within the range from 0.39 to 0.91 among the various ethnic groups, whereas the 174M allele – from 0.08 to 0.14. In the present study 235T and T174M allele frequencies were 0.15 and 0.16 in our control population, respectively.
The results of our study provide evidence of an association between the AGT gene variants and the risk for CHD in the Rostov population. In agreement with several other studies that have detected the AGT gene polymorphisms role in the development of this disease in other ethnic groups, including Spanish population (15), Slovaks (16), Polish (17) and Tunisians (18) etc., we ascertained that the homozygosity for 235T was associated with increased risk for CHD in Rostov population. However, it was revealed that M235T variant as well as T174M do not contribute to the increased risk of CHD in the Indian population.(11)
Table 3: The allele and genotype frequencies of the AGT M235T and T174M polymorphisms in various populations |
Population |
Sample size (n) |
AGT M235T |
AGT T174M |
References |
Genotype frequency |
Allele frequency |
Genotype frequency |
Allele frequency |
MM |
MT |
TT |
M235 |
235T |
TT |
TM |
MM |
T174 |
174M |
Japanese (Nagoya) |
352 |
3.7% |
31.8% |
64.5% |
0.20 |
0.80 |
82.7% |
16.2% |
1.1% |
0.91 |
0.09 |
(22) |
Japanese (Oita) |
170 |
9.4% |
25.3% |
65.3% |
0.22 |
0.78 |
80.0% |
18.8% |
1.2% |
0.89 |
0.11 |
(20) |
Chinese (Taiwan) |
337/336* |
1.2% |
16.0% |
82.8% |
0.09 |
0.91 |
80.4% |
19.0% |
0.6% |
0.90 |
0.10 |
(23) |
Chinese (Shanghai) |
90 |
7.8% |
24.4% |
67.8% |
0.20 |
0.80 |
81.1% |
18.9% |
0% |
0.91 |
0.09 |
(24) |
Indian
(India) |
131 |
8.4% |
30.5% |
61.1% |
0.24 |
0.76 |
77.9% |
20.6% |
1.5% |
0.88 |
0.12 |
(11) |
Bedouin (Gulf) |
61 |
26.2% |
42.6% |
31.1% |
0.47 |
0.53 |
77.0% |
21.3% |
1.6% |
0.88 |
0.12 |
(25) |
German (Berlin) |
102 |
38.2% |
45.1% |
16.7% |
0.61 |
0.39 |
78.4% |
20.6% |
1.0% |
0.89 |
0.11 |
(26) |
German (Giessen) |
511 |
32.9% |
48.3% |
18.8% |
0.57 |
0.43 |
76.1% |
22.5% |
1.4% |
0.87 |
0.13 |
(27) |
French and Irish |
741 |
34.8% |
50.2% |
15.0% |
0.60 |
0.40 |
78.0% |
20.8% |
1.2% |
0.88 |
0.12 |
(28) |
Austrian |
732/731** |
32.4% |
48.8% |
18.9% |
0.57 |
0.43 |
74.1% |
24.1% |
1.8% |
0.86 |
0.14 |
(29) |
Danish |
7975 |
34.9% |
48.7% |
16.4% |
0.59 |
0.41 |
77.1% |
21.4% |
1.4% |
0.88 |
0.12 |
(30) |
Italian (Florence) |
209 |
40.2% |
41.1% |
18.7% |
0.61 |
0.39 |
76.6% |
22.0% |
1.4% |
0.88 |
0.12 |
(31) |
Russian (Moscow) |
90 |
22.2% |
46.7% |
31.1% |
0.46 |
0.54 |
75.6% |
23.3% |
1.1% |
0.87 |
0.13 |
(32) |
Russian (Tomsk) |
122 |
- |
- |
- |
- |
- |
84% |
16% |
0% |
0.92 |
0.08 |
(33) |
Russian (Tomsk) |
150 |
30% |
42% |
25% |
0.52 |
0.48 |
- |
- |
- |
- |
- |
(34) |
*- the sample size differ for allele and genotype frequencies calculations for M235T and T174M polymorphisms (337 and 336, respectively);
** - the sample size differ for allele and genotype frequencies calculations for M235T and T174M polymorphisms (732 and 731, respectively). |
The lack of association for the T174M variant with CHD in our study appears to be in conflict with those reports implicating it in the disease.(18-20) It is possible that these controversial results point to the practical relevance of the ethnic variability, and could be explained by several factors, such as inter-ethnical variation.(21)
Mechanism by which AGT-235T genotype could be contributing to the development of CHD is unknown. In most cases the association of the AGT gene variants with different cardiovascular diseases could be illustrated by direct influence of the AGT on the blood pressure level. Other factors involved in artery wall injury should be considered.
Conclusion:
In conclusion, the results indicated the significant associations of the AGT gene polymorphism M235T with CHD risk in the Rostov population. However, further gene-gene and gene-environment interactions investigations are required to study the associations. Furthermore genetic association studies involving very large sample size are needed to provide conclusive evidence on the effects of the AGT gene and other genes within the RAS system on risk of CHD.
Funding:
This research was supported by projects of the Ministry of Education and Science of Russia №6.703.2014/K. Analytical work was carried out on the equipment of the Center for collective use of Southern Federal University «High Technology», grant 14.594.21.0002.
References
- Demographical yearbook of Russian Federation. Official edition 2013 // Russian Federal Statistics Service (Rosstat). - ISBN 978-5-89476-295-1. – M. – 2013. – 525 p.
- O'Toole TE, Conklin DJ, Bhatnagar A. Environmental risk factors for heart disease. Rev Environ Health. 2008 Jul-Sep;23(3):167-202.
- Cooney MT, Kotseva K, Dudina A, et al. Determinants of risk factor control in subjects with coronary heart disease: a report from the EUROASPIRE III investigators. Eur J Prev Cardiol. 2013 Aug;20(4):686-91.
- Tan ST, Scott W, Panoulas V, et al. Coronary heart disease in Indian Asians. Glob Cardiol Sci Pract. 2014;2014(1):13-23.
- Pranavchand R, Reddy BM. Current status of understanding of the genetic etiology of coronary heart disease. J Postgrad Med. 2013 Jan-Mar;59(1):30-41.
- Hinohara K, Ohtani H, Nakajima T, et al. Validation of eight genetic risk factors in East Asian populations replicated the association of BRAP with coronary artery disease. J Hum Genet. 2009 Nov;54(11):642-6.
- Ellis KL, Palmer BR, Frampton CM, et al. Genetic variation in the renin-angiotensin-aldosterone system is associated with cardiovascular risk factors and early mortality in established coronary heart disease. J Hum Hypertens. 2013 Apr;27(4):237-44.
- Liang B, Qin L, Wei H, et al. AGT M235T polymorphisms and ischemic stroke risk: a meta-analysis. J Neurol Sci. 2013 Aug 15;331(1-2):118-25.
- Zafarmand MH, van der Schouw YT, Grobbee DE, et al. The M235T polymorphism in the AGT gene and CHD risk: evidence of a Hardy-Weinberg equilibrium violation and publication bias in a meta-analysis. PLoS One. 2008;3(6):e2533.
- Sui X, Gao C. The angiotensinogen gene M235T polymorphism and acute myocardial infarction risk: a meta-analysis of 22 studies. Mol Biol Rep. 2013 Jul;40(7):4439-45.
- Nair KG, Shalia KK, Ashavaid TF, et al. Coronary heart disease, hypertension, and angiotensinogen gene variants in Indian population. J Clin Lab Anal. 2003;17(5):141-6.
- Miller SA, Dykes DD, Polesky HF. A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res. 1988 Feb 11;16(3):1215.
- Rodriguez S, Gaunt TR, Day IN. Hardy-Weinberg equilibrium testing of biological ascertainment for Mendelian randomization studies. Am J Epidemiol. 2009 Feb 15;169(4):505-14.
- Petrie A, Bulman JS, Osborn JF. Further statistics in dentistry Part 8: Systematic reviews and meta-analyses. Br Dent J. 2003 Jan 25;194(2):73-8.
- Rodriguez-Perez JC, Rodriguez-Esparragon F, Hernandez-Perera O, et al. Association of angiotensinogen M235T and A(-6)G gene polymorphisms with coronary heart disease with independence of essential hypertension: the PROCAGENE study. Prospective Cardiac Gene. J Am Coll Cardiol. 2001 May;37(6):1536-42.
- Jurkovicova D, Sedlakova B, Riecansky I, et al. Cardiovascular diseases and molecular variants of the renin-angiotensin system components in Slovak population. Gen Physiol Biophys. 2007 Mar;26(1):27-32.
- Buraczynska M, Pijanowski Z, Spasiewicz D, et al. Renin-angiotensin system gene polymorphisms: assessment of the risk of coronary heart disease. Kardiol Pol. 2003 Jan;58(1):1-9.
- Abboud N, Ghazouani L, Kaabi B, et al. Evaluation of the contribution of renin angiotensin system polymorphisms to the risk of coronary artery disease among Tunisians. Genet Test Mol Biomarkers. 2010 Oct;14(5):661-6.
- Li X, Li Q, Wang Y, et al. AGT gene polymorphisms (M235T, T174M) are associated with coronary heart disease in a Chinese population. J Renin Angiotensin Aldosterone Syst. 2013 Dec;14(4):354-9.
- Cong ND, Hamaguchi K, Saikawa T, et al. A polymorphism of angiotensinogen gene codon 174 and coronary artery disease in Japanese subjects. Am J Med Sci. 1998 Nov;316(5):339-44.
- Ludwig EH, Borecki IB, Ellison RC, Folsom AR, Heiss G, Higgins M, et al. Associations between candidate loci angiotensin-converting enzyme and angiotensinogen with coronary heart disease and myocardial infarction: the NHLBI Family Heart Study. Ann Epidemiol. 1997 Jan;7(1):3-12.
- Ichihara S, Yokota M, Fujimura T, et al. Lack of association between variants of the angiotensinogen gene and the risk of coronary artery disease in middle-aged Japanese men. Am Heart J. 1997 Aug;134(2 Pt 1):260-5.
- Ko YL, Ko YS, Wang SM, et al. Angiotensinogen and angiotensin-I converting enzyme gene polymorphisms and the risk of coronary artery disease in Chinese. Hum Genet. 1997 Aug;100(2):210-4.
- Liu Y, Jin W, Jiang ZW, et al. Correlation between T174M and M235T polymorphism of angiotensinogen gene and coronary heart disease. J Diagn Concepts Pract. 2002 ;1:231–6.
- Frossard PM, Hill SH, Elshahat YI, et al. Associations of angiotensinogen gene mutations with hypertension and myocardial infarction in a gulf population. Clin Genet. 1998 Oct;54(4):285-93.
- Wenzel K, Blackburn A, Ernst M, et al. Relationship of polymorphisms in the renin-angiotensin system and in E-selectin of patients with early severe coronary heart disease. J Mol Med (Berl). 1997 Jan;75(1):57-61.
- Gardemann A, Stricker J, Humme J, Nguyen QD, Katz N, Philipp M, et al. Angiotensinogen T174M and M235T gene polymorphisms are associated with the extent of coronary atherosclerosis. Atherosclerosis. 1999 Aug;145(2):309-14.
- Tiret L, Ricard S, Poirier O, et al. Genetic variation at the angiotensinogen locus in relation to high blood pressure and myocardial infarction: the ECTIM Study. J Hypertens. 1995 Mar;13(3):311-7.
- Renner W, Nauck M, Winkelmann BR, et al. Association of angiotensinogen haplotypes with angiotensinogen levels but not with blood pressure or coronary artery disease: the Ludwigshafen Risk and Cardiovascular Health Study. J Mol Med (Berl). 2005 Mar;83(3):235-9.
- Sethi AA, Tybjaerg-Hansen A, Gronholdt ML, et al. Angiotensinogen mutations and risk for ischemic heart disease, myocardial infarction, and ischemic cerebrovascular disease. Six case-control studies from the Copenhagen City Heart Study. Ann Intern Med. 2001 May 15;134(10):941-54.
- Fatini C, Abbate R, Pepe G, et al. Searching for a better assessment of the individual coronary risk profile. The role of angiotensin-converting enzyme, angiotensin II type 1 receptor and angiotensinogen gene polymorphisms. Eur Heart J. 2000 Apr;21(8):633-8.
- Babunova NB, Minushkina LO, Zateishchikov DA, et al. [Association of the T174M and M235T polymorphisms of the angiotensinogen gene with myocardial ischemia in the Russian population of the city of Moscow]. Mol Biol (Mosk). 2003 Jan-Feb;37(1):57-60.
- Spiridonova MG, Stepanov VA, Puzyrev VP, et al. The estimation of gametic disequilibrium between DNA markers in candidate genes for coronary artery disease (CAD) and the associations of gene complexes with risk factors for CAD. Int J Circumpolar Health. 2001 Apr;60(2):222-7.
- Pavlova KK, Trifonova EA, Edacheva AA, et al. Molecular genetic analysis of M235T angiotensinogen gene marker in the populations of different ethnic origin during gestosis. Siberian journal of Medicine. 2011;26(3):74-8.
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