Differential expression analysis of coronary heart disease related genes in Hainan residents

ZHANG Shun-li, XIE Cai-chan, LIAO Lu-xiao, CHEN Yong-kun, ZHANG Shi-long,WANG Xiao-qi

1.Department of Cardiovascular Surgery, The Second Affiliated Hospital of Hainan Medical University, Haikou 570311, China

2.Department of Cardiology, Yunnan Fuwai Cardiovascular Disease Hospital, Kunming 650000, China

Keywords:

ABSTRACT Objective: To explore the correlation between coronary heart disease related genes and coronary heart disease in hospitalized patients in Hainan, and to provide theoretical basis for enriching the screening methods of high-risk groups of coronary heart disease in Hainan,and optimizing the prevention and treatment strategies.Methods: We select hospitalized patients born in Hainan and aged >30 years old from the Second Affiliated Hospital of Hainan Medical Unversity between January 1, 2020 and June 30, 2022, and divided the patients into the coronary heart disease group and the non-coronary heart disease group.PCR real-time fluorescence was used to measure gene expression, and Spearman correlation analysis was used to explore the correlation between gene expression and coronary heart disease.Results: A total of 55 whole blood samples were collected from non-coronary heart disease patients (including 26 women and 29 men), with a median age of 57 years, and 170 whole blood samples from coronary heart disease patients (including 44 women and 126 men), with a median age of 63.17.Apolipoprotein B gene (ApoB) was highly expressed in patients with coronary heart disease (P<0.001); AGT gene (P=0.015 8), ApoE gene (P=0.012 6), FGB gene (P=0.005),GNB gene (P=0.015 1), MTFHR gene (P=0.011 9), SEL gene (P=0.005), TNF gene (P=0.029 8) were significantly overexpressed in the non-coronary heart disease group.The expression of NOS3 gene (P=0.304 7), IL6 gene (P=0.723 9), ACE gene (P=0.785 2)was not different between the two groups.Coronary heart disease was negatively correlated with AGT gene(r=-0.163, P=0.011, P<0.05), positively correlated with APOB gene(r=0.75, P=0, P<0.01),negatively correlated with FGB gene(r=-0.163, P=0.011, P<0.05), negatively correlated with GNB gene(r=-0.165, P=0.011, P<0.05), negatively correlated withSEL gene(r=-0.171,P=0.007, P<0.01), negatively correlated with MHTHR gene(r=-0.210, P=0.001, P<0.01) and negatively correlated with TNF gene(r=-0.131, P=0.04, P<0.05), but coronary heart disease was not correlated with APOE, NOS3, ACE, IL6 and other genes (P>0.05).The ApoB gene of coronary heart disease was negatively correlated with triglyceride (r=-0.461, P=0), positively correlated with age (r=0.173, P=0.009), positively correlated with total cholesterol (r=0.499,P=0), negatively correlated with high-density lipoprotein (r=-0.181, P=0.007), and negatively correlated with low-density lipoprotein (r=-0.143, P=0.031).Conclusion: (1) The detection of apolipoprotein B gene expression may be used as an indicator for screening coronary heart disease in the coronary population in Hainan.It may increase the risk of coronary heart disease by affecting the level of total cholesterol.A larger sample of research is still needed.(2)AGT,ApoE, FGB, GNB, MTFHR, SELE, TNF and other genes may be the protective genes of non-coronary heart disease population in Hainan, and the high expression of these genes may reduce the occurrence of coronary heart disease, which still needs further study.

1.Introduction

With the rapid economic development of the country, morbidity of coronary heart disease (CHD) has also ramped up obviously, the age of onset is gradually getting younger, and CHD has become one of the major causes of death over the world.CHD has many complications, such as angina pectoris, arrhythmia, heart failure,thromboembolism, myocardial infarction, and cardio-arrest.It is difficult to treat coronary heart disease, and neither drug treatment nor surgical treatment can cure it.Surgery is currently the most effective treatment for coronary artery disease.however, one study found that the mortality rate among coronary patients treated with PCI was 1.6% and the rate of revascularization was 5.9% at 1-year follow-up; the mortality rate among patients with coronary artery bypass grafting was 0.9% and the rate of revascularization was 3.9%[1].According to the Global Health Estimates 2020 report published by the World Health Group, the number of global deaths from cardiovascular disease in 2019 was 55.4 million, and coronary heart disease deaths accounted for 16% of global deaths[2].The American Heart Association’s Heart Disease and Stroke Statistics 2020 update reported 18.2 million people with coronary heart disease in the United States in 2016 among adults aged 20 years and older, with an overall prevalence of 6.7%[3].As of 2018, heart disease became the most deadly disease of all in the U.S.The number of deaths from coronary heart disease was 365,914 in 2017, with a mortality rate of 92.9%[4, 5].According to the China Cardiovascular Health and Disease Report 2021[6] reported that cardiovascular disease in China is on a continuous rise and is currently estimated to be about 330 million with 11.39 million coronary heart disease, with a mortality rate of 121.59/100,000 for urban residents and 130.14/100,000 for rural areas in 2019.the total cost of hospitalization for cardiovascular disease in China in 2019 was 313.366 billion yuan.Among them, the total hospitalization cost of cardiovascular diseases was 177.338 billion yuan, of which 125.625 billion yuan was for ischemic cardiomyopathy, and repeated treatments imposed a huge economic burden on patients.After years of research on coronary heart disease, it has been clearly established that coronary heart disease is a complex multifactorial disease,including environmental and genetic factors.The Framingham study has confirmed a series of risk factors for coronary heart disease, including age, sex, family history, smoking, hypertension,dyslipidemia, and diabetes, which are called “traditional risk factors”for coronary heart disease “ [7, 8].Genome-wide association analysis studies (GWASs) conducted by several research teams worldwide on coronary heart disease susceptibility genes in different populations have identified 161 coronary heart disease susceptibility genes/loci and have strongly contributed to the development of coronary heart disease genetics[9-12].A study of more than 20 000 Swedish twins confirmed the increased risk of coronary heart disease (CHD)between close relatives and estimated the heritability of fatal CHD to be approximately 50%; another analysis quantifying heritability using a genome-wide association study approach similarly estimated the heritability of CHD to be 40 - 50%[13].

Converting enzyme gene (ACE), Guanine nucleotide binding protein subunit β three gene (GNB3), Methylenetetrahydrofolate reductase gene (MTH reductase gene (MTHFR), endothelial nitric oxide synthase gene (NOS3), E-selectin gene (ESLE),interleukin-6 gene (ESLE), and the Interleukin-6 gene (IL-6), tumor necrosis factor gene (TNF), and plasma fibrinogen B gene (FGB)are considered to be associated with coronary artery disease and contribute to the development and disease progression of coronary artery disease, and are These coronary heart disease-related genes are expected to be the targets of coronary heart disease treatment in the future.

As the research on coronary heart disease deepens, the importance of early prevention of coronary heart disease onset is increasingly highlighted.Recently, “zero-level prevention” has been introduced by domestic scholars mainly in the field of cardiovascular disease.The term “zero and prevention” was first introduced in 1978 by Toma Strasser, a foreign scholar, who believed that measures should be taken to prevent the emergence of risk factors for cardiovascular disease, thereby preventing the prevalence of risk factors in the population and thus preventing the occurrence of cardiovascular disease.The concept of “zero-level prevention” is centered on the health of the person, focusing on the prevention and control of health or disease risk factors before they occur, emphasizing the prevention and control of cardiovascular disease from “before a new life is born,” “before the risk is present “This is consistent with the core concept of “treating the disease before it occurs” in Chinese medicine[14].Since there are individual, racial and geographical differences in the expression of coronary heart disease-related genes,an in-depth understanding of regional coronary heart disease gene expression can help to provide a scientific method for the screening of coronary heart disease high-risk groups in the region, which is conducive to the zero-level prevention of coronary heart disease in the region.As China is a unified multi-ethnic country, the expression of coronary heart disease genes may differ among ethnic groups.Therefore, we selected 11 genes that are recognized at home and abroad as being associated with coronary heart disease and contain traditional risk factors for coronary heart disease, and studied their expression in Hainan, in order to provide Hainan residents with a method for early screening of people at high risk of coronary heart disease, and to provide a method for the “zero-level prevention”of coronary heart disease in Hainan people.The study will provide a theoretical basis for “zero-level prevention” of coronary heart disease in Hainan, improve the quality of medical care, and enhance the capabilities of comprehensive prevention and control of coronary heart disease in Hainan.

2.Materials and methods

2.1 Study population

In this study, a case-control was used and inpatients who visited the Department of Cardiovascular Surgery and Department of Cardiology from the Second Affiliated Hospital of Hainan Medical College from January 1, 2020 to June 30, 2022 were selected, and the samples were of Hainan origin.The study is in accordance with the terms of the Declaration of Helsinki and has been approved by the Medical Ethics Committee of the Second Affiliated Hospital of Hainan Medical University.The relevant informed consent forms were signed with the patients.

2.1.1 Inclusion criteria

1) Adult inpatients attending the Department of Cardiac Surgery or Cardiology at the Second Affiliated Hospital of Hainan Medical University from January 1, 2020 to June 30, 2022.

2) Nationality is Hainan and have lived in the Hainan area for 30 years or more.

3) Completing coronary angiography during hospitalization.

2.1.2 Grouping criteria

According to the diagnostic criteria of coronary heart disease in the“Stable Coronary Heart Disease Primary Care Guidelines (2020)”,patients with coronary angiography results confirming subepicardial coronary artery diameter stenosis >50% and patients with typical angina symptoms or non-invasive tests suggesting myocardial ischemia diagnosed as coronary heart disease were classified in the coronary heart disease group; patients who did not meet this diagnostic criteria were classified in the non-coronary heart disease group.

2.1.3 Exclusion criteria

(1) Inpatients whose place of residence is not Hainan nationality excluded.

(2) Inpatients with severe heavy infections, liver and kidney insufficiency, tumor diseases, heart failure excluded, co-existing chronic infections rheumatic immune system diseases excluded.

2.2 Approach to the study

2.2.1 Collection of clinical data

Age, gender, history of hypertension, history of diabetes, history of smoking, history of alcohol consumption, triglycerides, LDL, HDL,and cholesterol were collected from all study subjects.

Smoking history: Smoking was defined as >1 cigarette per day for >1 year according to the 1984 WHO criteria for smoking, or the patient had smoked but had quit for

History of alcohol consumption: >100 g of alcohol per occasion,more than 2 times per week for more than 6 months.

2.2.2 Sample collection and processing

On the morning of the following day, the inpatient was admitted to the hospital on an empty stomach, the elbow vein was routinely disinfected, and peripheral venous blood was collected and sent to the hospital’s Laboratory Department (testing equipment: fully automated biochemical analyzer BS2000M Shenzhen Myri; fully automated biochemical instrument 7600-110 Hitachi) for the determination of TG, TG, HDL and LDL.3-5 mL of blood samples were also collected and placed in tubes containing anticoagulant and stored at a normal temperature.The blood samples were sent to Hainan Pratt Technology Co., Ltd.on the same day to complete relevant gene extraction and quantitative gene expression testing.

2.3 Statistical methods

The data were processed using SPSS statistical software (version 26) with a test level of α=0.05.General normal measures were described by mean ± standard deviation, non-normal measures by median M, and count data by rate and composition ratio.The t-test was used for measurement data conforming to normal distribution, non-parametric test was used for measurement data not conforming to normal distribution, and chi-square test was used for categorical data and count data.To explore the correlation between the expression of coronary heart disease genes and coronary heart disease and general clinical information using spearman correlation analysis; to explore the differential expression of coronary heart disease genes in the two groups; to explore the correlation between highly expressed genes and general clinical information; to explore the correlation between factors with differences and highly expressed genes in the coronary heart disease group; to explore the correlation between highly expressed genes in coronary heart disease and coronary heart disease combined with hypertension, diabetes mellitus, and family history of coronary heart disease The correlation between the gene expression and the family history of coronary heart disease.Gene expression was tested for significant differences using the Students t-test, with P.

3.Results

3.1 Clinical data

The number of cases in the coronary heart disease group (170) was higher than the number of cases in the non-coronary heart group(55), and in terms of gender, the number of men in the coronary heart disease group (126) was higher than the number of women in the non-coronary heart disease group (26), and the number of women in the non-coronary heart disease group (26) was lower than the number of men (29), and there was a difference in gender between the two groups, P=0.03, P< 0.05).Total cholesterol was higher in the coronary artery disease group than in the non-coronary artery disease, and the difference between the two groups was statistically significant (P=0, P< 0.05), shown in Table 1.

3.2 Relationship between coronary heart disease genes and coronary heart disease

It was found from Table 2 that CHD was negatively correlated with AGT gene (rs=-0.163, P=0.011, , P=0.011, P<0.05),APOB gene (rs=0.75, P=0, P<0.01), FGB gene (rs=-0.163, P=0.011,P<0.05), GNB gene (rs=- 0.165, P=0.011, P<0.05), SELE gene was negatively correlated (rs=-0.171, P=0.007, P<0.01), MHTHR gene was negatively correlated (rs=-0.210, P=0.001, P<0.01), TNF gene was negatively correlated (rs=-0.131, P=0.04, P<0.05 ), and no correlation with genes such as APOE, N0S3, ACE, and IL6(P>0.05), as shown in Table 2:

3.3 Expression of genes in both groups

Apolipoprotein B gene (ApoB) was highly expressed in the CHD group t=7.816 P<0.0001 (as in Figure 1A); AGT gene t=3.126 P=0.0020 (as in Figure 1B, ApoE gene t=3.641 P=0.0003 (as in Figure 1C), FGB gene t=3.384 P=0.0008 (as in Figure 1D), GNB gene t=3.337 P=0.001 (as in Figure 1E), MTFHR gene 3.937 P=0.0001 (as in Figure 1F), SELE gene t=3.132 P=0.0020 (as in Figure 1G), TNF gene t=2.585 P=0.0104 (as in Figure 1H) were highly expressed in the NCHD group; NOS3 gene t=1.719 P=0.0870 (as in Figure 1I), IL6 gene t=1.755 P=0.0806 (as in Figure 1J), and ACE gene t=0.5845 P=0.5595 (as in Figure:1K) were not statistically significant between the two groups, as shown in Figure1

Tab 1 Comparison of baseline data between the CHD and NCHD groups

Tab 2 Correlation between coronary heart disease gene expression and coronary heart disease (Spearman Rho)

3.4 Correlation analysis of highly expressed genes and blood lipids in CHD group

The ApoB gene for coronary artery disease was negatively correlated with triglycerides with a correlation coefficient (rs=-0.461,P=0), positively correlated with age with a correlation coefficient (rs=0.173, P=0.009), positively correlated with total cholesterol with a correlation coefficient (rs=0.499, P=0), negatively correlated with high-density lipoprotein with a correlation coefficient(rs=-0.181, P= 0.007), and negatively correlated with LDL with a correlation coefficient (rs=-0.143, P=0.031) as shown in Table 3.

Fig 1 Relative size of gene expression in two groups of patients

Tab 3 Association between apolipoprotein B gene and blood lipids(Spearman Rho)

4.Discussion

Because of the high incidence of coronary heart disease, many complications, incurable, unclear pathogenesis, high cost, and high economic burden for patients, the study of its mechanism,epidemiology, risk factors, and risk prediction genes, the search for early identification of coronary heart disease, early intervention,and optimization of coronary heart disease treatment have drawn scientific researchers from various countries to explore.Hainan province is located in the southernmost part of China and subtropical.Several studies in China have found ACE gene polymorphism, AGT gene polymorphism, acetaldehyde dehydrogenase 2(ALDH2) gene polymorphism, AOPE gene polymorphism, mutation of cholesterol transport protein (CETP) gene, Klotho gene polymorphism, E670G polymorphism of PCSK9 gene, sterol regulatory factor binding protein cleavage-activating protein (SCAP) gene isoleucine (Ile)796 valine (Val) polymorphism are associated with coronary heart disease (CHD) in the Hainan population and can increase the prevalence and risk of CHD[15-20].Previous studies have found a significant correlation between Apo B and lipid metabolism in blood,and the Apo B gene encodes apolipoproteins of celiac particles and LDL, which are the strongest predictors of coronary heart disease[21, 22].In this study, we found that the apolipoprotein B gene was positively correlated with coronary heart disease in patients with coronary heart disease in Hainan.

In this study, we found that AGT gene (P=0.0158), ApoE gene(P=0.0126), FGB gene (P=0.005), GNB gene (P=0.0151), MTFHR gene (P=0.0119), SELE gene (P=0.005) and TNF gene (P=0.0298)were highly expressed in the non-coronary group.The expression of ACE genes (P=0.7852) and others did not differ between the two groups, which is inconsistent with the findings of Yun Meiling et al[16], Zhou Daifeng et al[17], and Zhang Yong et al[15], analyzing the reasons as follows: (1) samples with stenosis in coronary arteries not meeting the diagnostic criteria for coronary artery disease by contrast examination were included in the non-coronary artery disease group, which makes the samples in the non-coronary artery disease group may also be potential coronary artery disease patients,which makes no statistical difference in gene expression between the two groups.Developing more detailed inclusion criteria might have yielded different results.(2) The sample size selected was small,especially in the non-coronary group, and increasing the sample size might have yielded different results.(3) Different populations have different lifestyles and are not exposed to the same risk factors.

The advantage of the results of this study is that the 11 genes related to coronary heart disease that are more recognized at home and abroad were studied at the same time, and the expression of these genes in each sample was determined by the mature PCR technique to explore the expression differences between the coronary heart disease and non-coronary heart disease populations in Hainan, and the whole experiment was measured by Hainan Pratt Technology Co.The sample size included in this study is not large enough, and there may be a selection bias, and the sample size should be expanded;2.There may be more complex relationships between genes and environment that have not been found, so the scope of influencing factors needs to be expanded for further study; 3.In this study, these genes do not play a role or play a very minor role in the development of coronary heart disease; 4.The phenotypes and polymorphisms of each gene in the coronary heart disease and non-coronary heart disease groups were not found.group and non-coronary group were not explored and could be continued in subsequent studies.

The advantage of the results of this study is that 11 genes related to coronary heart disease that are more recognized at home and abroad were studied at the same time, and the expression of these genes in each sample was determined by the mature PCR technique to explore the expression differences between coronary heart disease and non-coronary heart disease populations in Hainan, and the whole experiment was measured by Hainan Pratt Technology Co.The sample size included in this study is not large enough, and there may be a selection bias, and the sample size should be expanded;2.There may be more complex relationships between genes and the environment that have not been discovered, so the scope of influencing factors needs to be expanded for further study; 3.Among the genes in this study, these genes did not play a role in the development of coronary heart disease or played a very minor role;4.The phenotypes and polymorphisms of each gene in the coronary heart disease and non-coronary heart disease groups were not found.group and NCHD group were not explored and could be continued in subsequent studies.

5.Conclusion

1.Apolipoprotein B gene is a high-risk factor indicator for the coronary heart disease population in Hainan, potentially enriching the screening tools for the high-risk coronary heart disease population in Hainan and able to improve our current prevention strategies.

2.Genes such as AGT, ApoE, FGB, GNB, MTFHR, SELE, and TNF may be protective genes for coronary heart disease population in Hainan, and high expression of these genes may reduce the occurrence of coronary heart disease, further studies are still needed.5.Disclosure statement and author contributions

The article was written by Shunli Zhang; the article was revised and directed by Xiaoqi Wang; Yongkun Chen,Lu-Liang Liao and Shilong Zhang were responsible for data collection.

The authors have nothing to disclose.

Conflict of interest: none declared.