APOA5 gene polymorphism assessment and association with hypertriglyceridemia in the military force of the State of Goiás

According to the World Health Organization (WHO), dyslipidemias are determinant for the onset of cardiovascular diseases and represent more than 30% of global deaths. Changes in serum lipid levels are a direct consequence of dyslipidemia. This study aimed to verify the relationship between genetic polymorphism APOA5 and dyslipidemia. This is a case-control study with 199 officers of the military force of the state of Goiás in which the relationship between lipid profile and genetic polymorphism rs964184 was evaluated. Real-time Polymerase Chain Reaction (realtime PCR) was used to identify genetic polymorphism rs964184. Of the participants analyzed, 93% were male and 7% female, the lipid profile showed that 115 participants had dyslipidemia (group of cases) and 84 were within the reference range (control group). The genetic polymorphism rs964184 of the case group showed that 64.3% (74/115) of the participants had genotype CC, 33.0% (38/115) had cg genotype and 2.6% (3/115) had GG genotype. In the control group, 69.0% (58/84) had genotype CC, 27.4% (23/84) had cg genotype, 3.6% (3/84) had GG genotype. In conclusion, the G alllea is related to high levels of triglycerides (≥ 150 mg/dL) and the C allea is related to normal HDL cholesterol levels (≥ 40 mg/dL). Therefore, homozygous (GG) or heterozygous (CG) individuals are more inclined to have high triglycerides and low HDL cholesterol levels, which are associated with an increased risk of developing cardiovascular disease.


Introduction
According to the World Health Organization (WHO), cardiovascular diseases (CVD) are responsible for about 17.9 million deaths every year, among several risk factors, dyslipidemia is prominent in the development of CVDs (WHO, 2017).
Dyslipidemia is characterized as changes in serum lipid levels (ANVISA, 2011). According to the United States National Health and Nutrition Examination Survey (NHANES), about 53% of American adults have some lipid disorder (Tóth et al., 2012). In Brazil, according to research conducted in the state of São Paulo, the prevalence of dyslipidemia in the population of the state was 59.74% (Garcez et al., 2014).
Dyslipidemias can be classified as hyperlipidemias and hypolipidemias, being etiologically characterized as primary (genetic origin) or secondary causes (unhealthy lifestyle, morbid conditions, or even medications) (SBC, 2017).
According to the type of changes in serum lipid levels, dyslipidemia is classified as follows: a) Isolated hypercholesterolemia -an isolated increase in low-density lipoprotein cholesterol (LDL-c ≥ 160 mg / dL); b) Isolated hypertriglyceridemia -an isolated increase in triglycerides (TG ≥ 150 mg / dL or ≥ 175 mg / dL, if the sample is obtained without fasting); c) Mixed hyperlipidemia -an increase in LDL-c (LDL-c ≥ 160 mg / dL) and TG (TG ≥ 150 mg / dL or ≥ 175 mg / dL, if the sample is obtained without fasting). When TG is ≥ 400 mg / dL, the calculation of LDL-c by the Friedewald formula becomes inadequate, and mixed hyperlipidemia should be considered when high density non-cholesterol (HDL-c) is ≥ through polymorphisms, which can alter lipid metabolism (Andrade et al., 2002).
Regarding proteins involved in lipid metabolism, the apolipoproteins (apo) are characterized as the protein component of lipoproteins, participating in the formation of its five classes: chylomicron, High Density Lipoprotein (HDL), Low Density Lipoprotein (LDL), Intermediate Density Lipoprotein (IDL) and Very Low-Density Lipoproteins (VLDL) (Novak et al., 1996).
Apolipoprotein A5 (APOA5) has been identified as a new member of apolipoproteins, being located at locus 11q23 (van der Vliet et al., 2001) and plays an important role in mediating serum TG density. The findings related to APOA5 stimulated new research to determine the association of this genetic polymorphism with the plasma lipid levels in humans (Xia et al., 2015).
According to Genome-Wide Association Study (GWAS) multiple single nucleotide polymorphisms (SNP) associated with plasma TG levels have been identified. Among them, the SNP rs964184 stands out (chromosomal position: 11q23.3: 116648917), being close to the APOA5-A4-C3-A1 gene complex (ME 606368, ME 107690, ME 107720, ME 107680) (van de Woestijne et al., 2014). Several studies reported that the APOA5 T1131C polymorphism is related to an increased risk of cardiovascular disease in different ethnic groups (Xia et al., 2015).

Physiological changes caused by stress, homeostasis, and intermediate metabolism indicate that mental stress can lead
to the onset of cardiovascular diseases. In a stress situation, the human organism redistributes its energy sources, anticipating imminent aggression. This adaptation mechanism is advantageous if there is imminent danger. However, if this state persists for a long time, the damage will be inevitable. Stress can still provide an imbalance in the autonomic nervous system performance, triggering ischemic events and arrhythmias. (Loures et al., 2002).
In this context, this study aimed to assess the relation between APOA5 gene polymorphism and dyslipidemias, as well as the plasma changes in lipoproteins in military force officers of the State of Goiás.

Material and Methods
This study is part of the research project "Detection of genetic polymorphism for lipid alterations and alcohol use in Research, Society and Development, v. 10, n. 7, e8410716229, 2021 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v10i7.16229 Thus, the rs964184 polymorphism was genotyped, using the TaqMan Real Time PCR® kit (SNP Genotyping Kit, Applied Biosystems, USA), which contains the sense and antisense sequences of the primers that amplify the polymorphic sequence of interest and a Probe TaqMan® specific MGB allele that hybridizes a complementary sequence of the antisense primers. The fluorophores used will be FAM and HEX. Genotyping was performed by analyzing the fluorescence pattern of each sample on the StepOnePlus ™ systems thermocycler (Applied Biosystems, USA).
The PCR reactions were performed for a final volume of 15 µL containing 20 ng of genomic DNA, 2.5 µL of TaqMan® Universal Master Mix (R) (2X concentration), 0.25 µL of Custom TaqMan® Assay SNP Genotyping (20X concentrated) containing both primers and probes. The amplification protocol that was used followed the manufacturer's instructions, as described below: annealing of 60°C for 30s, followed by 95°C for 10 min and then 40 cycles containing denaturation of 95°C for 15s and annealing of 60 ° C for 1 min (Applied Biosystems, USA). This genetic analysis evaluated the polymorphism rs964184, which is associated with APOA5.
The serum samples were collected in 4 mL vacuum tubes with separating gel, which after centrifugation were used to perform the lipid profile, using enzymatic and direct methodologies in automated equipment A15 (Biosystems®), in the Clinical Laboratory of the Military Police Hospital of the State of Goiás. All quality assurance requirements have been met.
The samples were used only for the purpose of the study and at the end of the procedures, the samples were correctly disposed of according to RDC n° 306/2004 of the Agência Nacional de Vigilância Sanitária (ANVISA).
For the data analysis, descriptive statistics were performed, with the determination of the absolute and relative frequencies of the variables studied. Subsequently, inferential statistics were performed using Fisher's exact and G tests for categorical variables. The comparison of means and continuous variables were performed using Student's t-test or Mann-Whitney. Simple logistic regression was used to evaluate the polymorphisms in the groups, with the calculation of Odds Ratio (OR), the 95% confidence interval (95% CI), and the p-value. For all inferential tests, a significance level of 5% was adopted.
The statistical software used was BioEstat® 3.5.

Results
This was a case-control study, whose sample universe consisted of 200 active military police officers of similar ages.
From this total, one participant was classified as indeterminate, due to an inconclusive result in the SNP genetic polymorphism rs964184 evaluation, totaling 199 participants in this study. Most participants in the case group were over 40 years old, while the control group showed a balance (50%) between the ages. Dyslipidemia was observed in 115 individuals (case group), with the majority being evidenced in those who were overweight or obese. The highlighted lipid changes were the increase in total cholesterol and triglycerides and low HDL. Regarding the SNP rs964184, the CC genotype and the CG genotype were more commonly observed, while the GG genotype had a lower incidence (Table 1). The average age of the participants was 43.4 ± 8.5 years old for the case group and 39.8 ± 5.5 years old for the control group. Regarding the means of the continuous variables, there were significant differences in almost all variables, only the variable"height" did not show a significant difference (p =0.0966), according shown in Table 2. The rs964184 polymorphism showed to be related to dyslipidemia evidenced in the case group, among genotypes CC, CG, and GG. The highlighted variables were: increased levels in total cholesterol and triglycerides, and decreased levels in HDL cholesterol compared to the reference values (Table 3). Table 3. Absolute frequencies and relative percentages for the rs964184 polymorphism, 2020.

Control Case
Variable ( Table 4). Table 4. Absolute frequencies and relative percentages for the APOA5 rs964184 polymorphism, with the C allele dominant, 2020. The G allele dominant maintained the results when compared to the C allele dominant, the OR of triglycerides was very low demonstrating a greater chance of developing hypertriglyceridemia among the participants, while the HDL OR also presented a high value guaranteeing the protective factor (OR = 4.4615; CI95% = 2.091 -9.518) ( Table 5).

Discussion
Dyslipidemia is one of the main risk factors for the development of cardiovascular diseases (Moreira et al., 2006), the association between high levels of triglycerides and low levels of HDL cholesterol are common findings among patients with cardiovascular diseases. A similar association was observed in the case group assessed by this study.
Genetic factors are able to influence serum triglyceride levels, independently (van de Woestijne et al., 2014). The present study highlights that the polymorphism rs964184 of the APOA5 gene can directly influence the increase in triglycerides and total cholesterol. Similar results were reported by Woestijne et al. (2014), which observed a strong association of such polymorphism with triglyceride levels and a minor association with HDL-cholesterol levels.
Another study performed with the Chinese population (N = 5347) assessing the SNP rs964184, reported an association of polymorphism with increased triglyceride levels and significant relation between the six SNPs genotypes and the triglyceride levels (P < 0.008) (Fu et al., 2015).
Following the association of the rs964184 polymorphism with the risk of developing coronary heart disease, Xu and collaborators (2018), reported lower TG levels in older patients and suggested that these results were due to the use of drugs able to reduce this lipid rates in this age group. A similar result was observed in this study since lipid alterations were more associated with the population over 40 years old (69.6%).
The same study demonstrated that the male gender had a greater association with TG levels, significantly lower for men (p = 4.79 × 10-7) when compared to women (p = 0.716). The participants in the present study were predominantly men, with a total of 14 women, 7 belonging to the case group and 7 to the control group, whose results were similar.
This study demonstrated a greater association with the genotypes CG + GG and hypertriglyceridemia when compared to the CC genotype, being consistent with previous results performed in Iran and Tibet populations (Mirhafez et al., 2016;Parra et al., 2017;. Another observation about genotypes made by this study was the association of the G allele from SNP rs964184 with decreased levels of HDL, LDL, and total cholesterol, confirming the results previously reported . The polymorphism of rs964184 is directly related to high levels of triglycerides, which is a risk factor for the development of cardiovascular diseases. The application of polymorphism assessment can be used in nutrigenetics, which allows the detection of risks of these diseases through the genetic profile, demonstrating the necessity of changing habits in diet and lifestyle. Such fact could be observed in the present study, in which about 80% of the participants were overweight or obese, in addition to the change in lipid levels, and in this context, nutrigenetics could collaborate, as it is the study of the interaction between eating habits and the genetic makeup of individuals (Rimbach; Minihane, 2009).
Work tension may contribute to glucose disorders, justifying the higher risk of developing diabetes due to alcoholism, smoking, obesity, and sedentary lifestyle, but lipid levels or changes in pressure did not occur. The association between stress and coronary heart disease is mainly related to lifestyle and hyperglycemia. There are other potential biological stress factors to be analyzed such as chronic inflammation, blood-clotting factors, and increased risk of stress response that act as a stimulus of cardiac events (Nyberg et al., 2013).
The APOA5 gene is not the only one directly related to changes in TG levels, the APOE is another apolipoprotein that plays an important role in lipid metabolism that demonstrates a direct relationship in the increase of TG and consequently the increase in the risk of developing cardiovascular diseases (Demirag et al., 2007;Hagberg et al., 2000).

Conclusion
The APOA5 rs964184 gene polymorphism showed an important influence on increasing triglycerides levels and reducing the rate of HDL cholesterol, which is also related to the development of coronary diseases. The presence of the G allele in homozygosity or heterozygosity of the polymorphism rs964184 of the APOA5 gene was directly related to hypertriglyceridemia and decreasing HDL cholesterol, consequently, associated with a higher risk of developing cardiovascular diseases.
We recommend that there be future research based on apolipoproteins and cardiovascular diseases for possible treatments.