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Thrombosis risk factors and gene mutations in young age patients with acute coronary syndrome
https://doi.org/10.18087/cardio.2602
Abstract
Goal of research. Study the role of thrombosis risk factors and polymorphisms in genes in young age patients with acute coronary syndrome (ACS).
Materials and methods. Te study included 299 patients of age 25 to 44 years old with ACS were treated from 2012 to 2017 at the department of myocardial infarction 1st KGBUZ Altay regional cardiological clinic. Te middle age of patients with ACS was 40.3 ± 0.2 years. Te control group included of 53 apparently healthy volunteers aged from 25 to 44 years old, the average age those patients was 39.94 ± 0.79 years. Also, those patients hadn’t any comorbid conditions. Te control group hadn’t any datas of ischemic heart disease by the results of exercise tolerance tests. All patients had standard clinical, anamnestic, biochemical tests, lipid profle, fasting plasma glucose, electrocardiogram, echocardiography and coronaroangiography, also they were determined growth and weight with body-weight index. 116 patients from the ACS group and 53 patients fromthe control group had screening of polymerase chain reaction for determine polymorphism of the FII genes G20210-A, FV G1691-A, and MTHFR C677-T.
Results. We identifed the most signifcant sets of risk factors associated with ACS in young age patients based on our multifactorial statistical analysis with binary logistic regression. Tis combination of risk factors was: increased levels of low-density lipoproteins, decreased levels of high-density lipoproteins, smoking, existence of MTHFR homozygous polymorphism, heredity in combination with smoking, FV homozygote, MTHFR homozygote, smoking with MTHFR-homozygote.
Conclusion. Te ability predicting the risk of developing cardiovascular disease in young people based on traditional risk factors, partly modifable, as well as the researching of "new" risk factors, opens up new opportunities for developing a clinical approach of treating young patients with high risk of ACS.
For citations:
Ponomarenko I.V., Sukmanova I.A. Thrombosis risk factors and gene mutations in young age patients with acute coronary syndrome. Kardiologiia. 2019;59(1S):19-24. (In Russ.) https://doi.org/10.18087/cardio.2602
Cardiovascular diseases remain the main cause of premature death [1]. At present, an increasing rate of myocardial infarction (MI) is observed in young people of working age [2]. It is generally believed that the majority of patients who suffered MI at a young age has at least one identifiable cardiovascular risk factor [3, 4]. However, the conventional risk factors of atherosclerosis cannot fully explain the development of cardiovascular complications in young adults. A group of so-called "new" risk factors has been recently identified, which particularly include gene polymorphisms. The relationship between environmental factors and coronary artery disease (CAD) have been studied quite well, the value of genetic markers, by contrast, is not yet fully known [5]. Thus, for instance, according to the recent studies, there is a relationship between CAD and polymorphisms in genes encoding blood clotting factors, inhibitors of tissue plasminogen activator (PAI-I), fibrinolytic factors and platelet membrane receptors [5]. Special attention among a large number of candidate genes is attracted by polymorphic variants of blood clotting factor genes II (FII) and V (FV), a gene encoding methylenetetrahydrofolate reductase (MTHFR) (particularly its single nucleotide polymorphism S677T), which are associated with the development of cardiovascular diseases, which might be risk factors for acute coronary syndrome (ACS). However, data on the role of FII, FV, MTHFR gene polymorphisms in in the development of arterial diseases are controversial [6-8].
Thus, it is important to study risk factors and gene polymorphisms associated with the development of ACS in young patients for the improvement of early diagnosis, development and implementation of prevention programs in this patient population.
Objective was to study the role of conventional risk factors and gene polymorphisms in the development of ACS in young adult patients.
Materials and methods
This clinical study was approved by the Ethics Committee of Altai State Medical University, Russian Ministry of Health, and Altai Regional Cardiology Center. All patients signed the informed consent form before enrollment in the study. The study included 299 patients with confirmed ACS aged 25 to 44 years who were treated at the AMI Department of Altai Regional Cardiology Center from 2012 to 2017. The average age of the patients with ACS was 40.3±0.2 years.
Inclusion criteria:
1. Age 25–44 years old;
2. Confirmed diagnosis of MI with or without signs of atherosclerotic lesions of coronary arteries.
1. Age 25–44 years old;
2. Confirmed diagnosis of MI with or without signs of atherosclerotic lesions of coronary arteries.
Exclusion criteria: patients who refused to take part in the study, clinically significant comorbidities (noncoronary myocardial diseases, chronic comorbidities in the acute phase or incomplete remission, systemic diseases, acute inflammations, cancers).
The control group included 53 healthy volunteers aged 25 to 44 years (mean 39,94±0,79 years). Stress testing provided no data confirming CAD in the control group. The conventional risk factors were evaluated, and the standard general clinical and biochemical examinations with measurement of lipid metabolism and fasting glucose were performed in all patients. Besides 12-lead electrocardiography, all patients underwent echocardiography at Toshiba UTSHI9C system, coronary angiography using GeneralElectric device to determine the extent of coronary artery lesions and the possibility of percutaneous coronary intervention, height and weight were measured with calculation of body mass index (BMI). Polymorphisms in the FII G20210A, FV G1691A, MTHFR C677TAmpli genes were evaluated in 116 patients in the ACS group and 53 people in the control group. Genomic DNA was isolated from thawed venous blood by standard phenol-chloroform extraction method. Genotyping was performed by real-time polymerase chain reaction method by allelic discrimination using TaqMan probes, CFX96 amplifier (Bio-Rad), and Syntol primers and probes.
The statistical processing of the results was performed using the Statistica 10Rus and MS Excel 2007 software. Continuous variables are presented as M±m, where M is a sample mean and m is a standard error of mean. The samples were compared using a Student's t-test in case of a normal distribution and equality of sample variances. The linked samples were compared using a paired Student's t-test. If distributions are not normal, and dispersions are unequal, nonparametric Mann-Whitney U-test was used. Pearson's χ2 test was used to compare the frequency rates of attributes. Binary logistic regression analysis was used to identify risk factors for ACS. The significance level for a null hypothesis test was taken equal to p<0.05.
Results
In the study group, 230 (76.9%) patients were diagnosed with acute MI, 69 (23,1%) patients — with unstable angina. 141 (61%) patients had ST elevation MI (STEMI), and 89 (39%) patients had Non-ST elevation MI (NSTEMI).
In the STEMI subgroup, 56 (39.7%) patients underwent thrombolytic therapy, 112 (79.4%) patients underwent primary percutaneous coronary intervention (PCI). Pharmacoinvasive strategy was implemented in 54 (38%) patients. In 85 (53.8%) NSTEMI patients, NSTEMI was performed within the first 24 hours. A total of 197 (65.9%) PCI procedures was performed in ACS patients. Coronary angiography detected atherosclerotic coronary lesions in 265 (88.6%) patients, 20 (6.7%) patients had no atherosclerotic lesions of the coronary bed. Angiography revealed much less thrombosis — 12 (4%) cases, and only 2 (0.7%) cases of infarct-related artery spasm were diagnosed.
For 5 years, among 299 young adult patients hospitalized with ACS the majority of 259 (86.6%) patients were male, which was 6 times more than the number of women — 40 (13.4%). Patients with ACS and healthy volunteers did not differ in the frequency of a family history of early cardiovascular diseases (CVD) (30% and 28.3%, respectively, p=0.920). There were more smokers in the group of ACS patients, than in the control group (74.5% and 32%, respectively, p<0,001). Patients in the study group had higher BMI (29,0±0,52 and 25.96±0.56 kg/m2, respectively, p=0,001). More than half (65.6%) of young adult patients with ACS were overweight, there were less overweight patients in the control group (35,8%, p<0,001). 89 (29.7%) patients with ACS had grade I obesity, 20 (6,6%) — grade II obesity, 8 (2,6%) — grade III obesity; there were no obese patients in the control group. Despite the patients’ young age, the majority (n=191, 63.8%) had a history of hypertension: 55 (29%) patients in the study group had grade I hypertension, 48 (25%) patients had grade II hypertension, and 88 (46%) patients — grade III hypertension. In the ACS group, 34 (11.4%) patients had carbohydrate metabolic imbalance, of which the major portion was type 2 diabetes mellitus (DM) (n=28, 9.3%), type 1 diabetes (n=1,0 ,3%) was less common, impaired carbohydrate tolerance (n=1, 0.3%) and metabolic syndrome (n=4, 1.3%) (Guidelines on diabetes, pre-diabetes, and cardiovascular diseases, EASD/ESC, 2014). In the control group, no patients had disorders of carbohydrate metabolism. Mean glucose blood level was 6,06±0,13mmol/L in the ACS group, and 5.3±0.11 mmol/L in the control group (p=0.016).
Comparison of the lipid metabolism parameters revealed that ACS patients were diagnosed with hypercholesterolemia (119 (40%) and 10 (18.9%), p=0.006) and hypertriglyceridemia (174 (58%) and 7 (13.2%) p<0.001) more frequently than patients of the control group. Moreover, there were more patients with elevated levels of low-density lipoprotein (LDL) (132 (44%) and 8 (15.1%), p<0.001) and low levels of high-density lipoprotein (HDL) (128 (43%) and 6 (11%), p<0.001) in the study group, than in the control group. Patients with ACS had higher levels of total and LDL cholesterol, but lower levels of HDL cholesterol as compared with the control group (Table 1).
Table 1. Main indicators of lipid metabolism in ACS patients and healthy volunteers
Parameter | ACS group (n=299) | Control group (n=53) | p |
Total cholesterol, mmol/L | 6.29±0.19 | 4.44±0.12 | 0.001 |
Triglycerides, mmol/L | 2.35±0.10 | 1.88±0.31 | 0.085 |
LDL, mmol/L | 3.14±0.07 | 2.04±0.14 | 0.001 |
HDL, mmol/L | 0.97±0.02 | 1.27±0.06 | 0.001 |
On the next step, 116 patients with ACS and 53 volunteers were tested for genetic polymorphisms FII G20210A, FV G1691A, MTHFR C677T. In this study, we have decided to consider the FII 20210A, FV 1691A, MTHFR C677T alleles as mutant. Genotypes with mutant allele(s) were identified in 81 (69.8%) patients with ACS: one mutant genotype was found in 62 (53.4%) patients, two mutant genotypes — in 19 (16.3%) patients examined. In the control group, mutant genotypes were much rarer — 27 (50.9%) patients (p=0.028). In the ACS group, the most frequent polymorphism was MTHFR heterozygosity (C/T), no differences have been identified between the groups in frequency of this polymorphisms. MTHFR homozygosity (T/T) was less common, only 12% of patients with ACS and half of the healthy volunteers had this polymorphism. 17 (14.6%) patients with ACS had heterozygous FII polymorphism (G/A), and only 4 (3.4%) patients had FII homozygosity (A/A) . In the control group, no FII polymorphisms were found. In the ACS group, mutant genotypes of FV were presumably more frequent than those of the FII gene. Thus, FV heterozygosity (G/A) was detected in 18.9% of patients, which is more than in the non-ACS group. FV homozygosity (A/A) was detected in 9 (7.7%) patients with ACS, no homozygous FV polymorphisms were identified in the control group (Table 2).
Table 2. Results of the identification of genetic polymorphisms in ACS patients and healthy volunteers
Parameter | ACS group (n=116) | Control group (n=53) | p |
FII G20210A heterozygosity (G/A), n (%) | 17 (14.6) | 0 | 0.004 |
FII G20210A homozygosity (A/A), n (%) | 4 (3.4) | 0 | 0.310 |
FV G1691A heterozygosity (G/A), n (%) | 22 (18.9) | 2 (7.4) | 0.008 |
FV G1691A homozygosity (A/A), n (%) | 9 (7.7) | 0 | 0.058 |
MTHFRC677T heterozygosity (C/T), n (%) | 51 (43.9) | 12 (22.6) | 0.278 |
MTHFRC677T homozygosity (T/T), n (%) | 12 (12) | 14 (26.4) | 0.020 |
It is important to note, that mutant genotypes were identified in 83.3% of patients with thrombosis and without atherosclerotic coronary lesions.
In order to identify the most significant risk factors associated with ACS in patients examined, the method of statistical hypothesis test about equality of means and proportions to select the most significant factors which allowed determining significant differences between the group of young adults with ACS and the control group of healthy volunteers with null hypothesis probability of p<0.05. The following factor were selected: smoking (positive or negative smoking status), BMI, levels of total cholesterol, triglycerides, LDL and HDL; other risk factors were excluded due to lack of significant differences between patients with ACS and healthy volunteers.
The next step was to assess the relationship between the studied risk factors and the development of ACS, to this end we used the method of binary logistic regression to obtain parameters of the equation: y=a0+b1x1+b2x2+…+bnxn, where y is a value of the logistic regression function; а — empirical constant; x1…xn — values of predictors (independent variables); b1…bn — regression weighting factors calculated by logistic regression; n — number of predictors.
The analysis results showed that the studied factors have a statistically significant collective effect (χ2=41.1; p<0,001) on the classification results.
Table 3 shows the preliminary parameters of a logistic regression equation.
Table 3. Preliminary parameters of logistic regression analysis
Predictor included in the equation | Weighted regression coefficient (b) | Wald statistics (p) |
Empirical constant (a) | -1.998 | 0.235 |
BMI | 0.094 | 0.054 |
Total cholesterol | 0.031 | 0.896 |
TG | -0.104 | 0.519 |
LDL | 1.311 | <0.001 |
HDL | -2.067 | <0.001 |
Smoking status | -0.812 | <0.001 |
Thus, the examination of the young adult patients showed the risk factors with the most significant relationship with ACS were elevated LDL levels, low HDL levels, positive smoking status, while BMI, total cholesterol, triglycerides were excluded.
An independent analysis of the effect of genetic polymorphisms and their combinations with conventional risk factors on the risk of ACS development was carried out using binary logistic regression analysis. The following factors were selected: family history of early CVDs, positive smoking status, overweight, levels of total cholesterol, triglycerides, LDL, HDL, heterozygous or homozygous genotype for a mutant allele: FII G20210А, FV G1691А, MTHFR C677Т. 2 groups were studied: Group 1 (n=116) included patients with ACS with gene polymorphisms and Group 2 was the control group (n=53).
The parameters of a linear equation of the logistic function as described above. Table 4 shows the parameters of the logistic regression equation after the first step of analysis.
Table 4. Preliminary results of logistic regression analysis
Predictor included in the equation | Weighted regression coefficient (b) | Wald statistics (p) |
Empirical constant (a) | 15.27 | <0.001 |
FV-homozygosity | -0.29 | 0.611 |
MTHFR-homozygosity | 3.95 | <0.001 |
Heredity * Smoking | 1.73 | <0.001 |
Heredity * FV-homozygosity | -3.12 | <0.001 |
Smoking * FV-homozygosity | -3.51 | <0.001 |
Heredity * MTHFR-homozygosity | 3.38 | <0.001 |
Smoking * MTHFR-homozygosity | -1.07 | <0.001 |
Heredity * Smoking * MTHFR-homozygosity | 0.93 | 0.002 |
FV homozygosity had no effect on the development of ACS. The next step was a stepwise analysis excluding insignificant predictors from the estimations.
Discussion
Numerous studies have proven that male sex is an independent CAD risk factor, particularly in patients up to 45 years [4,9,10]. Indeed, 86.6% of hospitalized patients were male, which was 6 times more than female patients (13.4%). The frequency rate of early cardiovascular disease history was similar in patients with ACS and healthy volunteers. By contrast, the number of smokers among the patients with ACS was higher (74.5% and 32%, respectively). It has been proven that smoking is one of the most important modifiable risk factors in young patients with MI [5, 11]. Mean BMI in the ACS patients was higher than in the control group (29.0±0.52 vs 25.96±0.56 kg/m2, p=0.001). There were more overweight patients in the general group of patients than in the group of healthy people (65.5% vs 35.8%, p<0.001). Overweight and/or obesity are known to be independent predictors of MI [3, 10].
In the previous studies, a high prevalence of lipid imbalances in young patients with CAD as compared to the older age group [12]. All types of dyslipidemia were identified in the examined patients with ACS with almost the same rate. The analysis of the main parameters of lipid metabolism found that the levels of total cholesterol, LDL were higher in the ACS patients as compared to the controls (6,29±0,19 vs 4.44±0.12 mmol/L, p<0,001 and 3.14±0.07 vs 2.04±0.14 mmol/L, p<0,001). HDL cholesterol levels were lower than in the control group (0.97±0.02 vs 1.27±0.06 mmol/L, p<0.001). There were no significant differences between the groups in the triglyceride levels.
Along with well-known modifiable and non-modifiable risk factors, genetic thrombophilias were frequently identified in young adult patients (69.8%). There were statistically significant differences between the study groups in FII heterozygosity (17 (14.6%) vs 0%; p=0,004) and FV heterozygosity genotypes (18.9% vs 7.4%, p=0.008). No significant differences between the groups were identified in homozygous genotype, which may be explained by a small size of patient sample, thus, further studies in this direction are needed to provide additional data on associations of homozygous genotypes with the development of ACS.
Based on the multivariate analysis of the existing risk factors in young adult patients with ACS and using binary logistic regression, we determined the most significant risk factors associated with ACS. These risk factors were: elevated LDL levels (b=1,361; p<0.001), low HDL levels (b=–2,386; p<0.001), positive smoking status (b=0,830; p<0.001), existence of polymorphism in MTHFR — homozygous genotype (b=3,96; p<0.000), heredity in combination with smoking (b=1,73; p<0.000), heredity in combination with FV homozygosity (b=1,73; p<0.000), heredity in combination with MTHFR homozygosity (b=3,37; p<0.000), heredity in combination with smoking and MTHFR homozygosity (b=0,87; p<0.002).
Conclusions
Thus, along with well-known modifiable and non-modifiable risk factors, genetic thrombophilias were frequently identified in young adult patients with ACS. However, the role of gene polymorphism encoding components of the blood coagulation system in the increased risk of acute coronary syndrome has not yet been uniquely determined. Nevertheless, our findings showed several associations of conventional risk factors and gene polymorphisms with the development of ACS: The existence of homozygous MTHFR polymorphism, heredity in combination with smoking, heredity in combination with FV homozygosity, heredity in combination with MTHFR homozygosity, heredity in combination with smoking and MTHFR homozygosity. In this regard, it may be recommended along with the assessment of conventional risk factors to screen the young adult patients by testing FV G1691A, MTHFR C677T gene polymorphisms to form groups of high ACS risk, perform additional pre-clinical examinations (stress tests, duplex scanning of brachiocephalic arteries) and develop a program of preventive measures.
Further studies in this direction with larger samples are needed, which would allow us to learn more about the association of the above polymorphisms with the development of ACS.
No conflict of interests is reported
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About the Authors
I. V. Ponomarenko
Altai regional cardiological dispensary
Russian Federation
Russian Federation
Malakhov street 46, Barnaul, Altai Territory 656055
I. A. Sukmanova
Altai State Medical University
Russian Federation
Prospekt Lenina 40, Barnaul, Altai Territory 656038
Russian Federation
Prospekt Lenina 40, Barnaul, Altai Territory 656038
Review
For citations:
Ponomarenko I.V., Sukmanova I.A. Thrombosis risk factors and gene mutations in young age patients with acute coronary syndrome. Kardiologiia. 2019;59(1S):19-24. (In Russ.) https://doi.org/10.18087/cardio.2602
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