Frequencies and Distribution of APOE Gene Polymorphisms and Its Association With Lipid Parameters in the Senegalese Population

Background Apolipoprotein E is a multifunctional protein that plays an important role in lipid metabolism. It is encoded by the APOE gene. However, APOE gene polymorphism has not been very well studied in the Senegalese population. Therefore, we studied allele frequencies, genotype distributions, and the relationship between APOE gene polymorphisms and lipid parameters in the Senegalese women population. Methodology This study included 110 healthy women aged 35-72 years. The mean age was 49.8 ± 8.1 years. For all subjects, lipid parameters were analyzed from fasting serum, and APOE genotypes were identified by PCR-RFLP (polymerase chain reaction-restriction fragment length polymorphism) based analysis. Results Variations in the frequencies and distribution of the APOE alleles and genotypes were observed (ε3: 47.3%; ε2: 43.2%; ε4: 9.6%; and ε2/ε3: 70%; ε2/ε4: 16.4%; ε3/ε3: 10.9%; ε2/ε4: 2.7%). Compared to the ε3ε3 genotype carriers, carriers of the ε3ε4 genotype had a significantly higher rate of total cholesterol (p=0.03) and no high-density lipoprotein-cholesterol (p=0.02). Univariate analysis showed that the APOE ε4 allele increases the low-density lipoprotein-cholesterol rate (OR=3.06; 95% CI: 1.16-8.22; p=0.02). Conclusion Our study has shown a difference in APOE allele frequencies and genotype distributions with a total absence of ε2ε2 and ε4ε4 genotypes in a sample of Senegalese women. We also found that APOE gene polymorphism might play a role in plasma lipid levels.


Introduction
Apolipoprotein E (ApoE) is a polymorphic and multifunctional protein that is mainly synthesized by the liver. It is an essential apolipoprotein in plasma [1] and plays a vital role in the transport, metabolism, and digestion of lipoproteins [2][3][4]. ApoE promotes efficient clearance of circulating lipoproteins and participates in the cellular efflux of cholesterol [5]. ApoE plays an important role in regulating lipoprotein metabolism by regulating the bind of these lipoproteins to specific receptors. It can bind with different molecules, such as low-density lipoprotein-cholesterol (LDL-C), high-density lipoprotein-cholesterol (HDL-C), very lowdensity lipoprotein-cholesterol (VLDL-C), and chylomicron to participate in the transformation and metabolism of lipoprotein.
Therefore, the ε2, ε3, and ε4 genetic variants and their corresponding protein variations (E2, E3, and E4) have been linked to differential risks of dyslipidemia [11]. Dyslipidemia further confers as a risk factor of atherosclerosis, cerebral infarction, diabetes, and hypertension. Preventing dyslipidemia plays an important role in reducing morbi-mortality rate in the world [12]. At present, there are limited studies on the Senegalese populations. Hence, in the present study, we examined the allelic and genotypic frequencies of the APOE gene and its role in the lipid parameters in Senegalese women subjects firstly. Inclusion criteria were as follows: ≥ 18 years of age, females, and having not taken lipid-regulating drugs before or having stopped taking the drug for at least three months. Subjects were excluded if they were diagnosed with a disease that affected blood lipid level. Pregnant and breastfeeding women were also excluded.

Study participants and protocol
All procedures were conducted in accordance with the standards of the Declaration of Helsinki. It was reviewed and approved by the Ethics Committee of UCAD (Reference: Protocole 027512018/CERruCAD). All study participants provided signed informed consent.

Lipid analysis
Approximately 5 mL of fasting venous blood samples (at least 12 hours of overnight fasting) was collected into vacutainers from every participant and stored at -80°C in a deep freezer until further analysis.
All biochemistry parameters were analyzed on an automated Abbott device (ARCHITECT i1000SR, Abbott Laboratories, Abbott Park, Seattle, WA, USA) according to the standard laboratory protocol. The serum samples were assayed for blood lipid profiles. On the heparin tube, we measured the following lipids: apolipoprotein A (Apo A) apolipoprotein B (Apo B), total cholesterol (TC), HDL-C, LDL-C, and triglycerides (TG). TC, HDL-C, and TG were measured by the enzymatic method. Apo A and Apo B were measured by enzyme immunology.

DNA Extraction
Genomic DNA was extracted from peripheral blood lymphocytes using a commercial kit (REF A1125, Wizard® Genomic DNA Purification kit, Promega Corporation, Madison, WI, USA), as per the manufacturer's instructions. The DNA concentration was quantified using a NanoDrop 2000™ spectrophotometer (ThermoFisher Scientific, Waltham, MA, USA).

PCR-RFLP based analysis
APOE gene was amplified by polymerase chain reaction (PCR) on ThermoCycler (T-personal, Biometra, Jena, Germany). The PCR conditions included initial denaturation phase at 95°C for 5 minutes followed by 35 cycles of denaturation at 95°C for 30 seconds, annealing at 70°C for 20 seconds, extension phase at 72°C for 20 seconds, and the final elongation at 72°C for 10 minutes. PCR amplification was visually confirmed on 1.5% agarose gel electrophoresis.
After confirmation of amplification, 10 µl of each amplified DNA was mixed with each of the two restrictions enzymes (HaeII and AfLIII). The two reactions were allowed to proceed for at least 3 hours at 37˚C. The resulting fragments were analyzed on a 4% agarose gel ( Figure 1).

Statistical analysis
The data were expressed as mean ± SD and percentages (%). Allele frequencies were estimated by the gene counting method, and the chi-square test was used to identify departure from Hardy-Weinberg equilibrium. Strength of association between different lipid variables and APOE genotypes and alleles was estimated using THE chi-square test and logistic regression. SPSS statistical software Version 23.5 (IBM Corp., Armonk, NY, USA) was used for data analysis. A p-value of ≤0.05 was considered as significant in statistical analysis.

APOE alleles and genotype frequencies
We found that the ε3 allele is the predominant form while the ε2ε3 genotype is the most common form in the study population. In addition, there is a total absence for ε2ε2 and ε4ε4 ( Table 1).

Comparison of lipid profile between participant subgroups according to APOE gene polymorphisms
The ε3ε4 genotype compared to the ε3ε3 genotype had a significantly higher mean level of TC and no-HDL-C ( Table 2). We also found that only ε4 carriers had abnormal mean levels of total and LDL-C and that the mean TG level is normal regardless of the genotype considered.  We also found that carriers of the ε4 allele had higher blood LDL-C levels ( Table 3).

Discussion
ApoE, one of the major protein components of lipoproteins, plays an essential role in the circulation and the metabolism of blood lipids [13]. It is encoded by a gene that is characterized by polymorphisms. At present, there are limited studies on APOE gene polymorphism in the Senegalese population. Correlation between allelic and genotypic frequencies of APOE polymorphisms versus the risk of dyslipidemia served as a key attraction to conduct this study.
In this study, the analysis showed the three alleles of the APOE gene, which are all present in the Senegalese population with frequency distribution as ε3(47.3%)>ε2(43.2%)>ε4(9.6%). Furthermore, It should be mentioned that the APOE genotype distribution in this study was ε2ε3(70%)>ε2ε4(16.4%)>ε3ε3(10.9%)>ε3ε4(2.7%). However, some genotypes such as ε2ε2 and ε4ε4 were totally absent in our study population of adult subjects. We estimate that the absence of the ε2ε2 and ε4ε4 genotypes, in our study population consisting of elderly subjects, would be justified by a relatively short life expectancy for individuals harboring these genotypes. In effect, ε2ε2 homozygosity can precipitate type III hyperlipoproteinemia [14] while ε4ε4 would increase plasma LDL levels and the risk for atherosclerosis [2]. This differs from the results of some studies conducted in other populations such as in Algeria [15], where all APOE genotypes were present, and in another study from Saudi Arabia, researchers noted a total absence of ε2 and consequently the absence of the corresponding genotypes ε2ε3, ε2ε4, and ε2ε2 [16].
In the study, APOE ε2ε3 is the most common genotype and ε3 is the most predominant allele. This is contrary to previous studies because the ε3 allele is generally the most predominant allele but the most common genotype was ε3ε3 [17,18], and the worldwide frequency of the ε2, ε3, and ε4 alleles is 8.4%, 77.9%, and 13.7%, respectively [19]. Authors have reported that the ε2 isoform is the least common [15], which does not support our results, although the frequencies of APOE alleles and genotypes vary considerably between different ethnicities and populations [20].
In our study, we found that individuals harboring ε4 allele has higher blood LDL-cholesterol levels (OR=3.06; 95% CI: 1.16-8.22; p=0.02). In addition, the study shows that the individuals harboring ε3ε4 genotype compared to the individuals harboring ε3ε3 genotype had significantly a higher means levels of TC (p=0.03) and no-HDL-C (p=0.02). Our results are in line with the published studies. In the literature data, the ε4 isoform was associated with an increase in the TC and LDL-cholesterol concentrations when the ε3/ε3 homozygote carriers are used as references. Similar results have been observed in different studies [21,22]. Our study has shown a significant association between ε2 allele and the plasma TG levels (OR=1.05; 95% CI: 1.00-1.09). Several previous studies rather reported increased plasma TG levels in individuals harboring allele ε2 in healthy populations [23][24][25] and that TG concentration was significantly lower in the carrier of ε3 allele than in the carriers of ε2 or ε4 allele [21].
ApoE isoforms differ in their binding affinity to serum cholesterol and hence in their ability to remove dietary fats from the blood [26]. Accordingly, total serum cholesterol levels differ between APOE alleles and genotypes [26,27]. In view of these findings, it appears that despite differences of only one or two amino acids, the structural and functional differences between the three apoE isoforms may have a profound effect on disease risk [28].  [2]. In ApoE3, Arg-158 forms a salt bridge with aspartic acid-154, whereas in ApoE4, with Cys-158, this salt bridge is interrupted and aspartic acid-154 interacts with Arg-150, modifying the whole region receptor binding [2]. ApoE2 isoform shows defective binding to hepatic lipoprotein receptors [28]. Thus, the differential abilities of ApoE isoforms to bind to hepatic lipid receptors may contribute to ApoE isoformspecific effects in disease. This was confirmed by the study of Eto et al., which showed that the E2 allele and the E4 allele are associated with an increased risk of ischemic heart disease compared to the E3 allele [29].
The limits of our work are imputed to the study population in view of its small number and its exclusive composition of women. This study focused exclusively on a female population. In fact, in the literature, it is demonstrated that obesity is associated with polymorphisms of the APOE gene. In view of the variations in the prevalence of obesity according to gender, we wanted to first conduct a study in a female population and then, if possible, in an exclusively male population.

Conclusions
Our study has shown a significant difference in APOE allele frequencies and genotype distributions in the Senegalese women compared to other populations. The total absence of ε2ε2 and ε4ε4 genotypes in this Senegalese adult population requires a resumption to work on a younger cohort to better establish the results. We also found in the study that APOE gene polymorphism might play a role in determining plasma lipid levels.

Additional Information Disclosures
Human subjects: Consent was obtained or waived by all participants in this study. Institutional Ethics Committee of UCAD issued approval Protocole 027512018/CERruCAD. Animal subjects: All authors have confirmed that this study did not involve animal subjects or tissue. Conflicts of interest: In compliance with the ICMJE uniform disclosure form, all authors declare the following: Payment/services info: All authors have declared that no financial support was received from any organization for the submitted work. Financial relationships: All authors have declared that they have no financial relationships at present or within the previous three years with any organizations that might have an interest in the submitted work. Other relationships: All authors have declared that there are no other relationships or activities that could appear to have influenced the submitted work.