Aldehyde dehydrogenase 2 rs671 a/A Genotype is Associated with an Increased Risk of Early Onset Coronary Artery Stenosis

Introduction

Cardiovascular disease (CVD) is one of the major causes of disease burden and even death.¹ Atherosclerosis refers to the deposition of lipid and complex carbohydrate substances in the intima of the arteries, accompanied by the proliferation of fiber tissue and the deposition of calcium salts, which can lead to a large class of cardiovascular diseases.^2,3 Atherosclerotic cardiovascular diseases (ASCVD) include acute coronary syndrome (ACS), myocardial infarction (MI), stable or unstable angina pectoris, ischemic stroke, transient ischemic attack (TIA), and peripheral artery disease (PAD), which increase the risk for cardiovascular events.⁴ Atherosclerosis can lead to artery stenosis.^5,6 Coronary artery stenosis (CAS) is the formation of atherosclerotic plaque in the coronary arteries, resulting in the narrowing of the coronary arteries. There is a complex hemodynamic relationship between coronary stenosis and functional myocardial blood supply. In general, CAS often causes myocardial ischemia.⁵ The Third Report of the Adult Treatment Panel National Cholesterol Education Program (NCEP-ATP III) guideline defines early onset (premature) coronary heart disease as patients with coronary heart disease whose onset age is ≤55 years in men and ≤65 years in women.⁷ Predicting the risk of CAS is important for the prevention and treatment of CAS, especially the early onset CAS.

Aldehyde dehydrogenases can metabolize toxic aldehydes into low-toxic carboxylates, thereby reducing cell and tissue damage caused by oxidative stress.^8,9 Among all kinds of aldehyde dehydrogenases, aldehyde dehydrogenase 2 (ALDH2) is particularly important.^10,11 More and more studies have shown that ALDH2 is closely related to cardiovascular diseases such as atherosclerosis, hypertension, and cardiac insufficiency.^12,13 However, the relationship between ALDH2 and CAS risk is poorly understood. ALDH2 is encoded by the ALDH2 gene, which is located on chr12q24 and contains 13 exons.¹⁴ ALDH2 activity in vivo is influenced by ALDH2 gene polymorphisms.¹⁵ ALDH2 single nucleotide polymorphism (SNP) rs671 (G>A) is one of the most studied and important polymorphisms in ALDH2 gene. The change of A leads to the change of 504 amino acids from glutamic acid to lysine (Glu504Lys), and the activity of ALDH2 enzyme is reduced, or even completely lost. The Glu504Lys polymorphism can lead to a 30–50% enzyme activity of ALDH2.^16,17

There are not many studies on the relationship between ALDH2 polymorphisms and vascular stenosis. Qu et al found that ALDH2 rs671 A allele is a protective factor severe intracranial vascular stenosis in Han Chinese.¹⁸ Sung et al found that ALDH2 polymorphism was not associated with cerebrovascular stenosis and stroke functional outcomes.¹⁹ To date, there have been no reports on the relationship between ALDH2 polymorphisms and the risk of early onset CAS. In the present study, ALDH2 rs671 G˃A allele/genotype frequencies and the association between ALDH2 rs671 and the risk of early onset CAS patients were analyzed.

Materials and Methods

Study Participants

This study was a case-control study. 213 patients with early-onset CAS were enrolled as the study group, and 352 non-CAS patients of the same age group were enrolled as the control group from November 2019 to April 2023 in Meizhou People’s Hospital, Guangdong Province, China. CAS is diagnosed by coronary computed tomography angiography.⁵ Inclusion criteria of patients: (1) patients were diagnosed with CAS; (2) men with ≤55 years old and women with ≤65 years old; (3) the clinical data of the cases were complete. The inclusion criteria of the control group were: (1) non-CAS individuals who had been tested for ALDH2 rs671 polymorphism; (2) complete demographic information. Exclusion criteria: (1) congenital heart disease, cardiomyopathy, or congestive heart failure; (2) with severe organ dysfunction; (3) with malignant tumor; (4) with serious infectious diseases. Basic information collected and included in the analysis included age, sex, body mass index, smoking history, and alcohol abuse history. This study was performed in accordance with the ethical standards of the Declaration of Helsinki and approved by the Human Ethics Committees of Meizhou People’s Hospital. The flowchart of present study is shown in Figure 1.

Figure 1 The flowchart of this study.

Data Collection

Basic information was collected from our hospital’s medical record system, including age, gender, body mass index (BMI), history of smoking, and history of drinking. BMI was divided into three subgroups based on the Chinese criteria:^20,21 <18.5 kg/m², 18.5–23.9 kg/m², and ≥24.0 kg/m². Early morning fasting blood collection, serum separation. Lipid levels in serum samples were assessed using an automated biochemical analysis system (Olympus AU5400 system, Tokyo, Japan). The serum lipid-lipoprotein level measures include total cholesterol (TC), triglyceride (TG), high-density lipoprotein cholesterol (HDL), low-density lipoprotein cholesterol (LDL), Apolipoprotein A1 (Apo-A1), and Apolipoprotein B (ApoB).

DNA Isolation and ALDH2 Genotyping

Genomic DNA was extracted from venous blood collected from EDTA anticoagulant collection vessels using a blood DNA isolation kit (Qiagen GmbH, Germany). The quality and concentration of the DNA were assessed using a Nano-Drop 2000^™ spectrophotometer (ThermoFisher Scientific, Waltham, MA, USA). Polymerase chain reaction (PCR)-gene chip method was used for ALDH2 genotyping (BaiO Technology Co, Ltd., Shanghai, China). The PCR procedure was: 5 minutes at 94°C for initial denaturation, and 35 thermal cycles (94°C for 25s, 56°C for 25s, and 72°C for 25s). The products amplified by PCR were added into the gene chip, the amplification products of the wild-type template were hybridized with the wild-type probes, and the amplification products of the mutant template were hybridized with the mutant probes, and the genotype of the sample was determined by the hybridization reaction signal.

Statistical Analysis

Continuous variables were expressed as means ± standard deviations and were compared using either Student’s t-test or the Mann–Whitney U-test. Genotype composition ratios and allele frequencies between groups were analyzed with the Chi-square test. Hardy-Weinberg equilibrium in the patients and controls was evaluated by Chi-square test. Univariate analysis and multivariate regression logistic analysis were applied to examine the relationship between ALDH2 gene polymorphisms and early onset CAS. Multiple logistic regression analysis was performed after adjusting for gender, body mass index (BMI), history of smoking, history of drinking, and diabetes mellitus to assess the relationship between ALDH2 rs671 genotypes and early onset CAS risk. p<0.05 was considered to represent statistical significance. All statistical analysis were performed using SPSS statistical software version 26.0 (IBM Inc., USA).

Results

Characteristics of Subjects

There were 213 early onset CAS patients, and 352 controls in this study. The proportions of male and female in disease group and control group were 63.8% and 36.2%, 63.6% and 36.4%, respectively. There were 107 (30.4%) cases with BMI <18.5 kg/m² and 53 (15.1%) cases with BMI ≥24.0 kg/m² in controls, while 10 (4.7%) cases with BMI <18.5 kg/m² and 117 (54.9%) cases with BMI ≥24.0 kg/m² in patients. The proportion of overweight subjects in the patient group was much higher than that in the control group, the difference in BMI distribution among the groups was statistically significant (p<0.001). The proportion of smoking history, drinking history, and diabetes mellitus in patient group and control group were 27.7%, 5.6%, 37.1%, and 17.3%, 9.9%, 16.5%, respectively. The differences of TC (p<0.001), TG (p<0.001), HDL-C (p=0.006), LDL-C (p<0.001), Apo-A1 (p<0.001) and Apo-B (p<0.001) levels among the groups were statistically significant (Table 1).

Table 1 Clinical Characteristics of the Subjects of This Study

Distribution of the ALDH2 Genotypes and Alleles Between the Patients and Controls

The results of Hardy-Weinberg equilibrium test showed that the ALDH2 rs671 genotypes in the early onset CAS patients (χ²=0.871, p=0.351), and controls (χ²=0.727, p=0.394) confirmed to the Hardy-Weinberg equilibrium, respectively. Compared to the controls, the frequency of the ALDH2 rs671 G/G genotype was lower in the early onset CAS patients (43.7% vs 55.4%, p=0.007). The frequency of the ALDH2 rs671 G allele was lower (67.1% vs 75.0%) and A allele was higher (32.9% vs 25.0%) than that in the controls respectively, the difference in ALDH2 rs671 alleles distribution between the two groups was statistically significant (p=0.005) (Table 2).

Table 2 Distribution Frequencies of ALDH2 rs671 Genotype and Allele in Patients and Controls

Clinical Characteristics and Serum Lipid-Lipoprotein Levels of Subjects Stratified by ALDH2 rs671 Genotypes

The subjects were grouped according to ALDH2 rs671 genotypes, and the differences in clinical features and lipid levels were compared between the groups. ALDH2 rs671 G/G, G/A, and A/A genotype groups had statistically significant differences in the proportion of patients with a history of alcohol consumption (p=0.001). There were no statistically significant differences in the BMI distribution, history of smoking, diabetes mellitus, and serum lipid-lipoprotein levels among ALDH2 rs671 G/G, G/A, and A/A genotype groups (all p>0.05) (Table 3).

Table 3 Clinical Characteristics and Serum Lipid-Lipoprotein Levels of Subjects Stratified by ALDH2 rs671 Genotypes

Logistic Regression Analysis of Risk Factors Associated with Early Onset CAS Patients

Logistic regression analysis was used to evaluate independent factors of early onset CAS. Univariate regression analysis was performed to obtain the unadjusted odds ratio (OR), and multiple logistic regression analysis was performed to obtain the adjusted OR. Univariate logistic regression showed that ALDH2 rs671 G/A genotype (G/A vs G/G: OR 1.519, 95% confidence interval (CI): 1.064–2.171, p=0.022), A/A genotype (A/A vs G/G: OR 2.207, 95% CI: 1.124–4.334, p=0.021), overweight (BMI≥24.0 vs 18.5–23.9: OR 4.928, 95% CI: 3.262–7.443, p<0.001), history of smoking (yes vs no: OR 1.828, 95% CI: 1.216–2.748, p=0.004), and diabetes mellitus (yes vs no: OR 2.988, 95% CI: 2.012–4.439, p<0.001) were the risk factors of early onset CAS. After adjusting for other confounding factors, multivariate logistic regression showed that ALDH2 rs671 A/A genotype (A/A vs G/G: OR 2.508, 95% CI: 1.130–5.569, p=0.024), overweight (BMI≥24.0 vs 18.5–23.9: OR 5.047, 95% CI: 3.275–7.777, p<0.001), history of smoking (yes vs no: OR 2.813, 95% CI: 1.595–4.961, p<0.001), and diabetes mellitus (yes vs no: OR 2.191, 95% CI: 1.397–3.437, p=0.001) were the independent risk factors of early onset CAS (Table 4).

Table 4 Logistic Regression Analysis of Risk Factors for CAS

Discussion

CAS is a direct pathogenic factor of coronary artery disease (CAD), and the more severe the degree of CAS, the more likely it is to cause myocardial ischemia. Therefore, it is necessary to understand the risk factors leading to CAS. Previous studies mainly focused on the discussion of the risk factors of CAS in the general population and the elderly population.^22,23 This study investigated risk factors for early onset CAS and found that ALDH2 rs671 A/A genotype, overweight, smoking history, and diabetes mellitus were independent risk factors for early onset CAS.

SNP rs671 is the main polymorphic site of ALDH2 gene, which is related to the enzyme activity of ALDH2. Genotype G/G is a wild homozygote, and the enzyme activity encoded by this gene is normal. When the mutation occurs at this site, the spatial structure of the enzyme changes and the activity decreases, resulting in the deposition of aldehydes and easy damage to mitochondria.^24,25 The ALDH2 rs671 inactivation polymorphism is widely present in East Asian populations and is associated with an increased risk of cardiovascular disease.¹² Several studies have reported the relationship between ALDH2 gene polymorphism and CAD and coronary spastic angina (CSA). Xu et al found that people with ALDH2 gene mutation are more likely to develop coronary artery lesions than those with wild type.²⁶ Studies have shown that ALDH2 rs671 polymorphism may be a risk factor for CAD.^27,28 The study by Mizuno et al found that the ALDH2 re671A allele was a significant risk factor for CSA.²⁹ In addition, ALDH2 rs671A allele may be a protective factor for severe intracranial vascular stenosis in Han patients with ischemic stroke.¹⁸ However, the relationship between ALDH2 rs671 and CAS has not been reported before this study. The present study showed that individuals with ALDH2 rs671 A/A genotype increased risk of early onset CAS. This study enriched the data on the association between ALDH2 gene polymorphism and cardiovascular and cerebrovascular disease risk.

The mechanism of action of ALDH2 in the development of CAD is also being explored. The formation of foam cells plays an important role in the occurrence and development of atherosclerosis, Wei et al found that ALDH2 plays an important role in the formation of foam cells through the 4-HNE/PPARγ/CD36 pathway.³⁰ Another animal experiment showed that ALDH2 is also involved in the migration of coronary endothelial cells, which is involved in the process of coronary atherosclerosis.³¹ Yang et al found that ALDH2 may slow the progression of atherosclerosis by reducing endoplasmic reticulum stress and smooth muscle cell apoptosis.³²

Obesity can lead to the development of cardiovascular disease and can also directly contribute to the development of cardiovascular risk factors, including dyslipidemia, type 2 diabetes, and hypertension.^33,34 There is evidence that a higher body mass index is associated with an increased risk of most cardiovascular diseases.^35,36 The severity of CAS is higher in obese patients than in normal-weight patients.³⁷ Compared with normal patients, patients with obesity had lower coronary artery volume and myocardial mass ratio.³⁸ Of course, there are studies with different conclusions: BMI has no association with coronary atherosclerosis.^39,40 The mechanism by which obesity increases the risk of CAS include changes in body composition that affect hemodynamics and alter the microscopic structure of the coronary arteries.⁴¹ In addition, diabetes mellitus is also a risk factor for CAD.^42,43 Carlos Iribarren et al found that diabetes mellitus is an independent risk factor for early-onset CAD.⁴⁴ However, there have been no reports on the relationship between diabetes mellitus and the risk of early onset CAS.

Smoking is strongly associated with higher rates of CAD, heart failure, abdominal aortic aneurysm, ischemic stroke, transient ischemic attack, peripheral artery disease, and arterial hypertension.⁴⁵ Smokers have a higher risk of CAD severity.⁴⁶ A higher percentage of smokers was observed in patients with higher immature platelet fraction (IPF) values, smoking was an independent predictor of higher IPF values, and IPF was associated with cardiovascular disease.⁴⁷ And smoking is one of the predictors of CAS in Japanese men.⁴⁸ Majumder et al revealed that smoking is an independent risk factor for significant stenosis in coronary artery.⁴⁹ The mechanism of the role of smoking in the progression of atherosclerosis may be related to the effect of tobacco components on endothelial dysfunction, inflammation and lipid changes.⁵⁰

The present study showed that individuals with ALDH2 rs671 A/A genotype, overweight, smoking history, and diabetes mellitus increased risk of developing CAS among men ≤55 years old or women ≤65 years old. This study had some limitations. First, this case-control study is based on hospitalized patients and physical examination subjects, and selection bias of population is inevitable Second, in addition to analyzing the basic demographic information, smoking and drinking status and ALDH2 gene polymorphism of the subjects, this study did not collect and analyze other possible influencing factors, such as eating habits, sleep quality, and physical exercise status. Third, because the number of cases included in the current study is not enough, this study did not grade and compare the CAS patients according to the degree of CAS, but that’s one of the things we are working on.

Conclusion

In men ≤55 years old and women ≤65 years old, individuals with ALDH2 rs671 A/A genotype, overweight, smoking history, and diabetes mellitus increased risk of developing CAS. Revealing the characteristics and risk of early onset CAS is of great significance for the prevention and treatment of CAS.