Correlation between Apolipoprotein E genetic polymorphism and atrial fibrillation

Background We speculated that there was a correlation between apolipoprotein E (ApoE) genetic polymorphisms and the occurrence of atrial fibrillation (AF) based on the AF inflammatory mechanism. The high-risk alleles of ApoE were examined in patients with AF and controls to determine the distribution of genotype and allele frequencies. Methods From January 2017 to January 2019, 64 patients with AF in the department of cardiovascular medicine of the Lianyungang Second People's Hospital, and 49 healthy outpatient volunteers, were enrolled. ApoE gene polymorphisms were examined using allele-specific polymerase chain reaction. Statistical analyses were performed to identify high-risk ApoE alleles. Results A total of 113 patients were enrolled in this study. Among them, 64 patients were in the AF group (38 male and 26 female), with an average age of 74.38 ± 8.37 years. The control group consisted of 49 cases (29 male and 20 female), with an average age of 65.24 ± 12.14 years. The six ApoE phenotypes ε2/ε2, ε2/ ε3, ε2/ε4, ε3/ε3, ε3/ε4, and 4/ε4 were observed in 0.9% , 13.2%, 2.7%, 58.4% , 19.5%, and 5.3% . The proportions of our study population with ApoE protective, general, and risk genotypes accounted for 14.1%61.1% , and 24.8% , respectively. There was no statistically significant difference in ApoE gene polymorphism frequencies related to gender, height, weight, smoking status, hypertension, type 2 diabetes mellitus, and coronary heart disease (P0.05). There were significant differences in age, body mass indexBMI, larger left atrial diameterLAD, and left ventricle ejection fraction(LVEF) (P 0.05). The observed genotype frequencies were in Hardy–Weinberg equilibrium and were representative of the population. Conclusion There is a correlation between the ApoE genetic polymorphism and the occurrence of AF, and ApoEε4 is a high-risk genotype for AF.


Abstract
Background We speculated that there was a correlation between apolipoprotein E (ApoE) genetic polymorphisms and the occurrence of atrial fibrillation (AF) based on the AF inflammatory mechanism. The high-risk alleles of ApoE were examined in patients with AF and controls to determine the distribution of genotype and allele frequencies.
Methods From January 2017 to January 2019, 64 patients with AF in the department of cardiovascular medicine of the Lianyungang Second People's Hospital, and 49 healthy outpatient volunteers, were enrolled. ApoE gene polymorphisms were examined using allele-specific polymerase chain reaction. Statistical analyses were performed to identify high-risk ApoE alleles. Results A total of 113 patients were enrolled in this study.
Among them, 64 patients were in the AF group (38 male and 26 female), with an average age of 74.38 ± 8.37 years. The control group consisted of 49 cases (29 male and 20 female), with an average age of 65.24 ± 12.14 years. The six ApoE phenotypes ε2/ε2, ε2/ ε3, ε2/ε4, ε3/ε3, ε3/ε4, and 4/ε4 were observed in 0.9% , 13.2%, 2.7%, 58.4% , 19.5%, and 5.3% . The proportions of our study population with ApoE protective, general, and risk genotypes accounted for 14.1% 61.1% , and 24.8% , respectively. There was no statistically significant difference in ApoE gene polymorphism frequencies related to gender, height, weight, smoking status, hypertension, type 2 diabetes mellitus, and coronary heart disease (P 0.05). There were significant differences in age, body mass index BMI , larger left atrial diameter LAD , and left ventricle ejection fraction(LVEF) (P 0.05). The observed genotype frequencies were in Hardy-Weinberg equilibrium and were representative of the population. Conclusion There is a correlation between the ApoE genetic polymorphism and the occurrence of AF, and ApoEε4 is a high-risk genotype for AF. 3 Background Apolipoprotein E (ApoE) is plasma protein that plays an important role in regulating lipoprotein metabolism. ApoE is involved in lipid metabolism, oxidative stress, neuroimmunoregulation, and the inflammatory response [1][2] . There are three polymorphic ApoE alleles (ε 2, ε 3, and ε 4) and six genotypes can be formed [3] . Some of these genotypes may be susceptible to hyperlipoproteinemia and arteriosclerosis (AS) [4][5] . AS, cardiovascular, and cerebrovascular accident risk can be divided into three types with respect to ApoE. These are the ApoE protective (ε2/ε2 and ε2/ε3), popular (ε2/ε4 andε3/ ε3), and risk (ε3 /ε4,ε4/ε4) genotypes [6][7][8] .
Atrial Fibrillation (AF) is one of the most common cardiac arrhythmias seen clinically. The mechanism of AF is very complicated. AF is closely related to old age, smoking [9][10] , hypertension, diabetes, metabolic syndrome, dyslipidemia, being overweight or obese, and various cardiovascular diseases [11] . A large TARCS study in the United States found that elevated levels of lipids, especially LDL and cholesterol, increased the incidence of AF [12] . ApoE is involved in all aspects of blood lipid metabolism, including the synthesis, secretion, transport, and metabolism of lipoprotein, and has significant effects on blood lipid metabolism [13] . Therefore, it is likely that the ApoE gene is related to the occurrence of AF.
Inflammatory mediators including HS-CRP, IL-6, IL-8, TNF-α, TGF-β, and WBC are closely related to the occurrence and development of all types of AF [14][15][16] . An earlier study of myocardial biopsies in healthy individuals and patients with AF demonstrated that the irreversibility of AF is associated with myocardial remodeling due to persistent inflammatory infiltration of cardiomyocytes [17] . While ApoE has anti-inflammatory and anti-oxidative effects, ApoEε4 has a strong proinflammatory effect [18] and activates NF-κB.
Some studies show that NF-κB is involved in the process of atrial remodeling [19] . This further supports the hypothesis that ApoE is associated with AF. In this study, the association between ApoE genetic polymorphisms and AF was inferred based on the mechanisms of inflammation and lipid metabolism, and the ApoE allele representing a high AF risk was identified based on genotype and allele frequency distributions.

Patient population
Sixty-four patients (AF group) with AF (paroxysmal, persistent, long-term persistent, and permanent) and 49 healthy outpatients (control group) were randomly selected between January 2017 and January 2019 in the Department of Cardiovascular Medicine of the Lianyungang Second People's Hospital. There were 38 males and 26 females (mean age: 74.38 ± 8.37) in the AF group and 29 males and 20 females (mean age: 65.24 ± 12.14) in the control group.

Inclusion Criteria
The inclusion criteria were: clear ECG or Holter monitor data (absence of sinus P wave, presence of fast and irregular f wave, frequency 350-600 beats per minute, normal QRS complex shape and duration, and irregular R interval); clear clinical symptoms; a history of AF; and complete medical records and blood samples. This study was approved by the Medical Ethics Committee of the Second People's Hospital of Lianyungang (Grant no. L1618). All subjects signed an informed consent form.

Exclusion Criteria
The exclusion criteria were: serious infections; liver and kidney dysfunction; tumors; tuberculosis; metabolic syndrome; various major diseases affecting inflammatory factors and lipid metabolism; no complete medical history and blood samples; and failure to provide informed consent.

ApoE Polymorphism Test
Peripheral blood (4 ml) was extracted from fasting patients. The blood was extracted in the early morning into an EDTA anticoagulant blood vessel, and stored in the refrigerator at 4°C. All patient samples were sent to the unified medical examination center for ApoE gene polymorphism detection, and the test results were reported within 1 week.

Statistical analysis
All data were statistically analyzed using SPSS21.0 software. Measurement data was denoted by `x±s,and comparisons were made between two independent samples using an adapted t test. BMI measurements did not conform to normal distribution, and one-way ANOVA was used for comparison. The c2 test was used to compare the counting data.
Multivariate conditional logistic regression analysis was used to comprehensively evaluate the relationship between various factors and AF. P < 0.05 was considered statistically significant.

Discussion
ApoE is mainly expressed in the liver and brain and is an indispensable lipoprotein in physiological functions. The most important function of ApoE is to act as a ligand for the LDL receptor. After binding to the LDL receptor, ApoE enters liver cells for lipid metabolism and regulates lipid levels in the blood and brain. Anne et al. [20][21] reported that the occurrence and duration of AF may be related to electrical and structural remodeling induced by electro-mechanical feedback. Their results showed that ApoE activates NF-κB and induces the expression of monocyte chemoattractant protein-1 (MCP-1), TNF-α, IL-1β, and IL-10 [16] . In turn, MCP-1 induces IL-1, IL-6, and TNF-α expression in cardiomyocytes. This causes negative inotropic action in the heart, reduces heart function, and causes heart muscle structure reconstruction [24][25] . Furthermore, TNF-α may increase Ca 2+ concentration in cardiac myocytes [22] and the downregulation of L-type calcium (I Ca-L ) channels leads to the shortening of action potential duration and the effective refractory period, and the formation of multiple reentry loops. At the same time, Ca 2+ overload activates Ca 2+ -activated proteins, which can degrade cardiac contractile proteins such as troponin and lead to decreased atrial contractile function [23] . This can then cause atrial fibrosis, and further lead to abnormal intra-atrial impulse conduction and induced electrical remodeling of the atrium. This suggests an association between AF and the ApoE gene.
Our results showed no significant differences in sex, height, body mass, smoking status, hypertension, type 2 diabetes mellitus, and CHD between the AF and control groups (P 0.05). We also observed no sex difference in the ApoE gene frequency [26] . Significant differences in age, BMI, LAD, and LVEF (P 0.05) were observed between the two groups.
These results suggest that the risk of AF increases with increasing age. Moreover, these results indicate that higher BMIs lead to a larger LAD and lower ejection fraction (EF) and higher incidence of AF. Old age is an independent risk factor for AF [27][28] . People with a high BMI may have a greater amount of body fat, which increases the prevalence of AF.
The changes in LAD and EF caused by organic heart disease enlarges the atrium and changes its special structure. However, whether AF first affects the LAD or whether heart disease first increases the LAD, leading to AF, requires further research.
The distribution of the six ApoE phenotypes was uneven in the general population. The frequency of the ApoEε3 genotype was the highest, reaching more than 60%. Six phenotypes includingε2/ε2, ε2/ε3, ε2/ε4, ε3/ε3, ε3/ε4, andε4/ε4 were detected in 113 ApoE genotyping subjects. The frequency of ε3 /ε3 was the highest, ε3 /ε 4 was the second most common, and ε2 / ε2 was the least common. The distribution of the ApoEε3 genotype frequency in this study was 61.1%, accounting for more than half of the total. This was similar to that reported previously [29] . The risk of coronary heart disease, Alzheimer's disease, and stroke was normal for patients with the ApoEε3 phenotype, low for those with the ApoE ε2 phenotype, and highest for those with the ApoEε4 phenotype. In this study, subjects with ApoE protective, general and risk genotypes were measured in the two groups, accounting for 14.1%, 61.1% and 24.8%, respectively. Among them, subjects with ε 4 phenotype were 24.8%, which were high risk groups. Compared with those in the control group, there were slightly fewer patients with the ApoEε3 genotype and significantly more with the ApoEε4 genotype in the AF group. These results suggest that expression of the ApoEε4 genotype is related to AF.
Previous studies have shown that obesity is a risk factor for AF. This may be related to oxidative stress, inflammation, and other factors caused by abnormal lipid metabolism in obese individuals, which may lead to myocardial fibrosis atrial remodeling followed by AF [30] . Some studies suggest that ApoE ε4 is one cause of hyperlipidemia [31] . The association between obesity and hyperlipidemia may indirectly indicate that ApoE ε4 is a high risk factor for AF. If we can establish the relationship between the ApoE gene and AF and intervene at the gene level, it will be of great practical significance for the prevention and treatment of AF.
The disadvantages of this study are the limited sample size and the lack of large-scale and multi-region sampling. Moreover, all blood samples were tested externally, and the entire process of sample preservation, transportation, and detection was not able to be monitored.

Availability of data and materials
The data that support the findings of this study are available from The Second People's Hospital of Lianyungang.

Authors' contributions
WYL SLM were involved in the study concept and design. LHK WLQ DZ CRY TSJ were involved in the field study and data acquisition. WYL WML LHK were involved in the analysis and interpretation of the data.WYL WML LHK were involved in the drafting of manuscript. WYL SLM were involved in the critical revision of manuscript. All authors have read and approved the manuscript.

Ethics approval and consent to participate
This study was approved by the institutional review board ethical committee(The Second People's Hospital of Lianyungang Ethics Committee No L1618).

Consent for publication
Not applicable.

Competing interests
The authors declare they have no competing interests.