Non-lipid genetic predictors of the development of coronary heart disease and myocardial infarction in patients with hypertension

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BACKGROUND

Cardiovascular diseases (CVDs) are the leading cause of mortality among adults in the Russian Federation, accounting for 43.8% of the total number of lethal outcomes. In the structure of CVD-related mortality, coronary heart disease (CHD) represents 54.2%, making it an extremely pressing problem¹. Currently, more than 200 risk factors for CHD have been identified, including genetic factors. For a long time, the emphasis in the search for predictors of CHD was shifted toward lipid biomarkers. However, recent attention has shifted to non-lipid predictors of CHD, particularly low-grade inflammation, an important component of the cardiovascular continuum. Unsurprisingly, levels of pro-inflammatory markers correlate well with cardiovascular risk [1]. A meta-analysis by Y. Li et al. [2], combining results from 14 studies with 83,995 participants, showed a significant relationship between high-sensitivity C-reactive protein (CRP) and the risk of cardiovascular death and death from all causes. More modern works corroborate this relationship [3, 4]. Similar data have also been obtained for other proinflammatory agents. Thus, the STABILITY study, which included 14,611 patients, showed a clear association between the risk of developing major adverse cardiovascular complications and an increased (≥2 ng/l) level of interleukin-6 (IL-6) in blood serum [5]. This is also true for higher-order proinflammatory cytokines, such as IL-1, the main immunocompetent biomarker that can stimulate the production of both CRP and IL-6 [6].

Dysregulation of the Toll-like receptor (TLR) system may play a key role in maintaining chronic inflammation and progressing atherosclerosis. Several experimental studies on laboratory animals have convincingly demonstrated the significant role of TLRs in atherogenesis. For instance, a study by A. Kapelouzou et al. [7] demonstrated a high correlation between the expression of TLR types 2, 3, 4, and 8 and the progression of atherosclerosis in the aorta of male rabbits. Additionally, dysregulation of the hemostatic system contributes to the progression of atherosclerotic arterial lesions. Evidence shows that higher levels of plasminogen activator inhibitor 1 and homocysteine are associated with the formation of potentially more “hazardous” coronary plaques (atherosclerotic plaques with thin caps and large atherosclerotic plaques) [8].

However, several limitations prevent using these biomarker levels as reliable predictors of CHD and myocardial infarction (MI). For instance, the expression of inflammatory proteins may change due to persistent infection. Hence, the need arises to search for more reliable non-modifiable risk predictors, which may include genetic polymorphisms. Thus, a study by M. Asif et al. [9] revealed a correlation between the development of CHD and polymorphisms of the protein gene associated with the low-density lipoprotein receptor. The British Heart Foundation database contains 1,122 unique variants of low-density lipoprotein receptors, which are also associated with the development of CHD and are included in genetic screening for homozygous familial hypercholesterolemia (HFHC)². Similar studies are also available regarding the apolipoprotein B-100 protein [10].

There are also studies on polymorphisms of the proprotein convertase subtilisin/kexin type 9 (PCSK9) genes. Polymorphisms of the PCSK9 genes are known to enhance and inhibit its function. Thus, the T allele of single nucleotide polymorphism rs11206510, which increases its activity, is associated with a high risk of MI in CHD patients [11]. In turn, polymorphisms of the PCSK9 gene Y142X, C679X, and R46L protect the development of CHD and MI [12]. Low-density lipoprotein receptor adapter protein 1 (LDLRAP1) polymorphisms are rarer, namely, 432insA in exon 4 and G65A in exon 1 (p. trp22ter), and they are associated with the development of HFHC [13, 14].

Although there are publications regarding non-lipid genetic biomarkers of CHD, they are much fewer, and the results obtained are quite contradictory. This necessitates new studies to confirm or refute the contribution of any single nucleotide polymorphisms to the development of CHD and MI.

The study aimed to analyze the associations of the main non-lipid genomic biomarkers with the occurrence of CHD and the unstable course of the disease with the development of MI in patients with hypertensive disease (HD).

MATERIALS AND METHODS

The design was a retrospective cohort case-control study. It included 164 patients with stages I–III HD. The median age of patients was 67 (57–74) years; 90 (54.9%) of them were men. A total of 144 (87.8%) patients had dyslipoproteinemia, 101 patients (61.6%) had grades 1–2 abdominal obesity (AO), 84 (51.2%) patients were diagnosed with CHD, 52 (31.74%) patients had prediabetes (impaired glucose tolerance and/or impaired fasting glycemia), 34 (20.7%) patients had chronic kidney disease (CKD) stages C1–C3b. The gender, age, and clinical laboratory parameters of the patients included in the study are presented in Table 1.

 

Table 1. Gender, age and clinical laboratory parameters of patients included in the study at different stages, abs. (%)

Таблица 1. Половозрастные и клинико-лабораторные показатели пациентов, включенных в исследование на разных этапах, абс. (%)

Indicator	Stage 1		p	Stage 2		p
	CHD, n = 84	no CHD, n = 80		MI, n = 30	no MI, n = 54
Age, years	66	63.5	–	67	72.4	–
Male gender	38 (45.2)	52 (65)	0.01	17 (56.9)	21 (38.9)	0.21
Smoking	46 (54.8)	36 (45)	0.21	20 (66.7)	26 (48.1)	0.1
Dyslipoproteinemia	74 (88.1)	70 (87.5)	0.91	28 (93.3)	46 (85.1)	0.27
Prediabetes	34 (40.5)	18 (22.5)	0.01	12 (40)	22 (40.7)	0.95
CKD	15 (17.9)	19 (23.8)	0.35	7 (23.5)	8 (14.8)	0.33
Abdominal obesity	53 (63.1)	48 (60)	0.68	23 (76.7)	30 (55.6)	0.06

 

The study was conducted in two stages. At stage 1, a search was performed for non-lipid genetic predictors of CHD without taking into account the characteristics of its clinical course. For this purpose, all patients were distributed into two groups depending on the presence or absence of CHD. All patients underwent genetic typing of innate immunity (human leukocyte antigen class 2 (HLA-DRB1), TLRs type 3 (TLR3-412 L/F)), inflammatory cytokines (IL-6 (-174 C/G, rs1800795), IL-1b (-511 T/C, -1473 G/C), IL-10 (-1082 G/A, rs1800896), tumor necrosis factor α (-308 G/A), blood pressure regulatory systems (angiotensin-converting enzyme (ACE) (ACE Alu I/D, rs 4646994), angiotensin 2 (-235 M/T), hemostatic system (type 1 plasminogen activator inhibitor (SERPINE 1 (PAI-1)-675 5G/4G), coagulation factor 13 (F13A1-35 V/L), integrin beta 3 (ITGB3-33 L/P), folate cycle of methylenetetrahydrofolate reductase (MTHFR) -677 C/T, rs 1801133), methionine synthase reductase (MTRR-66 A/G, rs 1801394), and apoptosis inducers, namely, ataxia-telangiectasia mutation (ATM-185 Asp/Asn, rs 1801516). At stage 2, the possibility of identifying genetic predictors of the complicated course of CHD with the development of MI was studied.

Peripheral blood from patients was used for genetic typing and collected in ethylenediaminetetraacetic acid tubes upon discharge. Deoxyribonucleic acid (DNA) extraction was performed using the GS-Genetics kit, and DNA was amplified using the DT-Prime device from DNA Technology (Russia).

The study was conducted as part of the Virus research project and adhered to the provisions of the Declaration of Helsinki. It was approved by the ethics committee of the S.M. Kirov Military Medical Academy (Protocol No. 271, November 22, 2022). All patients signed informed, voluntary consent for medical intervention, personal data processing, and study participation.

When describing data for numerical normally distributed variables, the mean and standard deviation M (SD) were used, whereas the median and interquartile range Me [Q₁–Q₃] were used for non-Gaussian variables. Descriptive statistics used the number of occurrences of each value and the proportion of each value relative to the entire sample n (m%) for nominal and ordinal data. The search for differences was performed by testing statistical hypotheses. For normal samples, the Student’s t-test was used; for non-Gaussian samples, the Mann – Whitney U test was used. Nominal and ordinal samples were compared using the chi-square test, and Fisher's exact test was used for small samples (at least one of the expected values less than 10). In all hypothesis testing cases, the difference was considered statistically significant at p < 0.05. Calculations were performed in the statistical package R, version 4.3.1.

RESULTS AND DISCUSSION

It was established that the presence of CHD in HD patients was associated with a significant predominance of genetic biomarkers in only four single-nucleotide polymorphisms: the hemostatic system (4G4G SERPINE 1), proinflammatory cytokines (T allele IL-1b-511, C allele IL-1b-1473), and innate immunity (FF TLR3-412) (Fig. 1).

 

Fig. 1. Frequency of occurrence of genetic biomarkers in patients with hypertension associated with the development of coronary heart disease

Рис. 1. Частота встречаемости генетических биомаркеров у пациентов, страдающих ГБ, ассоциированных с развитием ИБС

 

The development of MI was associated with two genetic polymorphisms, namely, in the system of proinflammatory cytokines (CC IL-6-174) and hemostasis (4G4G SERPINE 1) (Fig. 2).

 

Fig. 2. Frequency of occurrence of genetic biomarkers in patients with stable ischemic heart disease and post-myocardial infarction

Рис. 2. Частота встречаемости генетических биомаркеров у пациентов со стабильным течением ИБС и перенесших ИМ

 

The association of genotype 4G4G polymorphism of the SERPINE 1 gene of the hemostasis system with a high risk of developing both CHD and MI in CHD patients was demonstrated. Thus, in the group of CHD patients, this genotype was significantly more common (40% vs. 9.5%; p = 0.007, odds ratio (OR) 11.4, 95% CI 2.24–89.2). A similar effect of this polymorphism was also demonstrated on the risk of MI occurrence. In the group of patients with a history of MI, it was significantly more common (66.7% vs. 20.7%; p = 0.026, OR = 14, 95% CI 1.85–296.1).

A meta-analysis by Y. Liu et al. [15], which included almost 150,000 people from different ethnic populations, demonstrated an association between the polymorphism of this gene and the development of atherosclerotic diseases. The study by A.L. Gonchar et al. [16] showed an almost twofold excess of the occurrence of the 4G4G genotype in patients with a history of MI (41.2% vs. 28.7%; p = 0.01). A more modern study by A. Bayramoglu [17] demonstrated the negative contribution of this genotype to the development of coronary atherosclerosis. This adverse effect may be attributed to the higher expression of tissue plasminogen activator type 1 in its carriers [18]. It has long been known that it is a potent antifibrinolytic enzyme [19]. In this regard, the progression of atherosclerotic lesions in the coronary vessels in carriers of this genotype can be due to the high potential for thrombogenesis resulting from the low activity of the antifibrinolytic system.

Our study also showed an association between polymorphisms of proinflammatory cytokine genes and the development of CHD and MI. Specifically, the T allele of the IL-1b-511 gene polymorphism was associated with the presence in CHD patients (60.9% vs. 36.9%; p = 0.033, OR = 2.66, 95% CI = 1.09–6.78). However, there are contradictory opinions regarding the association of this allele with the risk of developing CHD. For example, a meta-analysis by L. Zhou [20] found that this polymorphism has no association with the development and progression of atherosclerotic lesions of the coronary arteries. Conversely, the study by H. Rai [21] showed the protective function of the T allele and the negative function of the C allele of this gene. According to their data, carriage of the C allele significantly increases the risk of CHD (p = 0.041, OR = 1.36). Additionally, E.A. Zakharyan and O.Yu. Gritskevich [22] revealed an association between the TT genotype and the development of CHD in Caucasians. In their study, this genotype increased the risk of CHD by two times (OR = 2.2, 95% CI = 1.3–3.6). It is most logical to explain such heterogeneity in the results of researchers by ethnic differences in the studied groups of patients. All this again confirms the need for new research and reassessment of the contribution of this polymorphism to the development of cardiovascular pathology. An association with CHD was also revealed for the C allele of the IL-1b-1473 gene polymorphism. This allele was noted significantly more often in the group of CHD patients (60.9% vs. 34.9%; p = 0.021, OR = 2.9, 95% CI = 1.97–7.4). Currently, there is no convincing information about the effect of this polymorphism on cardiovascular pathology.

Our study also revealed a correlation of the CC genotype of the IL-6-174 C/G gene polymorphism, rs 1800795, with the development of MI in patients with stable CHD. This genotype was detected significantly more often in patients with a history of MI (26.5% vs. 2%, p = 0.004, OR = 25.7, 95% CI = 3.85–520.71). A number of studies have noted a similar correlation between the carriage of this genotype and the risk of developing thrombosis in various locations, including the coronary arterial bed. Thus, a meta-analysis of about 30.000 patients convincingly demonstrated the association of this genotype with the risk of MI and other arterial thrombosis [23]. This can be due to a higher level of IL-6 expression in carriers of this polymorphism [24].

We also established an association between polymorphism of the TLR gene, related to the innate immune system, and the occurrence of CHD. Thus, in the group of CHD patients, the FF genotype of the TLR3-412 gene polymorphism was significantly more common (25% vs. 3.2%; p = 0.046, OR = 10.33, 95% CI = 1.1–119.58). There are currently no studies regarding the contribution of this polymorphism to the course of CVDs, particularly CHD. However, a number of publications assess the association of this polymorphism with negative and protective effects in relation to a number of other nosologies. Currently, an association of this polymorphism with some autoimmune and oncological diseases has been established [25–28]. TLR3 is known to bind double-stranded ribonucleic acid (RNA) of viruses, bacteria, fungi, and messenger RNA isolated from necrotized cells [29].

An interesting study was conducted by G. Cooke et al. [30] demonstrated an association of the FF genotype with an enhanced proliferation potential of fibroblasts. This may well explain the progression of atherosclerotic lesions of the coronary arteries in patients carrying this genotype.

Overall, we have obtained data on the influence of the main non-lipid genetic biomarkers on the development of CHD and MI in patients with HD. CHD predictors include the 4G4G genotype of the SERPINE 1 gene polymorphism, the TT genotype of the IL-1b-511 gene polymorphism, the C allele of the IL-1b-1473 gene polymorphism, and the FF genotype of the TLR3-412 gene polymorphism. The negative impact of the 4G4G genotype of the SERPINE 1 gene polymorphism and the CC genotype of the IL-6-174 C/G, rs 1800795 gene polymorphism has been proven in relation to the risk of developing MI in individuals with a stable course of CHD. The use of these genetic biomarkers as predictors of poor prognosis in patients with HD will allow for better risk stratification and assessment of the prognosis in this group, which will significantly reduce the costs of conducting a genome-wide study.

CONCLUSIONS

Carriage of the 4G4G genotype of the SERPINE 1 gene, associated with the potential for hypercoagulation, increases the risk of CHD by four times.

Carriage of alleles and genotypes of proinflammatory cytokines associated with their higher expression also increases the risk of CHD and MI significantly more often, namely, the TT genotype of the IL-1b-511 gene polymorphism by more than two times, the C allele of the IL-1b-1473 gene polymorphism by almost three times. Carriage of the CC genotype of the IL-6-174 C/G gene polymorphism increases the risk of occurrence of MI in CHD patients by more than 10 times.

Polymorphisms of the TLR system genes are also associated with the risk of CHD occurrence. Thus, in the group of CHD patients, the FF genotype of the TLR3-412 gene polymorphism was eight times more common.

ADDITIONAL INFORMATION

Authors’ contribution. Thereby, all authors made a substantial contribution to the conception of the study, acquisition, analysis, interpretation of data for the work, drafting and revising the article, final approval of the version to be published and agree to be accountable for all aspects of the study.

The contribution of each author. M.S. Tyuryupov — development of the general concept, study design, article writing, data analysis; K.S. Shulenin — development of the general concept, study design, article writing, data analysis; T.S. Sveklina — review of publications, writing the article; T.G. Mirzoev — review of publications, writing the article; G.G. Kutelev — collection of clinical material, database compilation, statistical processing, article writing; D.V. Cherkashin — collection of clinical material, database compilation, statistical processing, article writing.

Competing interests. The authors declare that they have no competing interests.

Funding source. This study was not supported by any external sources of funding.

ДОПОЛНИТЕЛЬНАЯ ИНФОРМАЦИЯ

Вклад авторов. Все авторы внесли существенный вклад в разработку концепции, проведение исследования и подготовку статьи, прочли и одобрили финальную версию перед публикацией.

Вклад каждого автора. М.С. Тюрюпов — разработка общей концепции, дизайн исследования, написание статьи, анализ данных; К.С. Шуленин — разработка общей концепции, дизайн исследования, написание статьи, анализ данных; Т.С. Свеклина — обзор публикаций, написание статьи; Т.Г. Мирзоев — обзор публикаций, написание статьи; Г.Г. Кутелев — сбор клинического материала, составление базы данных, статистическая обработка, написание статьи; Д.В. Черкашин — сбор клинического материала, составление базы данных, статистическая обработка, написание статьи.

Конфликт интересов. Авторы декларируют отсутствие явных и потенциальных конфликтов интересов, связанных с публикацией настоящей статьи.

Источник финансирования. Авторы заявляют об отсутствии внешнего финансирования при проведении исследования.

 

¹ Demographic Yearbook of Russia [Internet]. URL: https://rosstat.gov.ru/storage/mediabank/Demogr_ejegod_2023.pdf (access date 03/17/2024).

² British Heart Foundation. LDLR Database. [Internet]. URL: http://www.ucl.ac.uk/ldlr/Current/summary.php?select_db=LDLR&show=sum. (access date 01/11/2024).

作者简介

Mark Tyuryupov

Kirov Military Medical Academy

编辑信件的主要联系方式.
Email: mark.tfyuryupov@icloud.com
ORCID iD: 0000-0002-8366-0594
SPIN 代码: 2886-7181

residency student

俄罗斯联邦, Saint Petersburg

Konstantin Shulenin

Kirov Military Medical Academy

Email: mark.tfyuryupov@icloud.com
ORCID iD: 0000-0002-3141-7111
SPIN 代码: 8476-1052

MD, Dr. Sci. (Med.), associate professor

俄罗斯联邦, Saint Petersburg

Dmitry Cherkashin

Kirov Military Medical Academy

Email: mark.tfyuryupov@icloud.com
ORCID iD: 0000-0003-1363-6860
SPIN 代码: 2781-9507

MD, Dr. Sci. (Med.), professor

俄罗斯联邦, Saint Petersburg

Tatyana Sveklina

Kirov Military Medical Academy

Email: sveklina@rambler.ru
ORCID iD: 0000-0001-9546-7049
SPIN 代码: 3561-6503

MD, Cand. Sci. (Med.), associate professor

俄罗斯联邦, Saint Petersburg

Gennady Kutelev

Kirov Military Medical Academy

Email: mark.tfyuryupov@icloud.com
ORCID iD: 0000-0002-6489-9938
SPIN 代码: 5139-8511

MD, Dr. Sci. (Med.)

俄罗斯联邦, Saint Petersburg

Nikita Mirzoev

Kirov Military Medical Academy

Email: mark.tfyuryupov@icloud.com
ORCID iD: 0000-0002-9232-6459
SPIN 代码: 9826-5624

residency student

俄罗斯联邦, Saint Petersburg

参考

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