Polymorphisms of APOE and APOB genes and dyslipidemias in a South Brazilian Cohort

Dyslipidemias are closely associated to the development of cardiovascular and cerebrovascular diseases, and therefore of great importance in public health. Many genes influence lipid levels, such as APOE and APOB . The present study aimed to investigate the effects of APOE gene alleles (rs7412: C>T and rs429358: T>C), and R3500Q mutation of APOB gene (rs5742904: G>A) in lipid levels in a South Brazilian cohort. 214 individuals were analyzed and the frequencies of APOE ε2 , ε3 and ε4 alleles were found respectively as 9.25 ± 0.4625; 70.5 ± 3.525 and 20.25%± 1.401, while the frequency of R3500Q mutation of the APOB gene was 4.46 ± 1.00%. The ε4 allele was significantly more frequent in subjects with higher TC (Total Cholesterol) and LDL-C levels, while significantly higher frequency of the ε2 allele was found in individuals with higher HDL-C levels. The R3500Q mutation was suggested as a risk factor for higher TC levels, regardless gender and age (β = 21.326 ± 10377.78, p = 0.001).


INTRODUCTION
Dyslipidemias constitute an important problem in public health, being related to the development of cardiovascular and cerebrovascular diseases (1,2,3).Around 53% of American adults have lipid abnormalities, including high levels of Low Density Lipoprotein Cholesterol (LDL-C) or Triglycerides (TG) and low concentrations of High Density Lipoprotein Cholesterol (HDL-C) (4).Coronary Arterial Disease (CAD) is considered the leading cause of death in Brazil and there is evidence that high cholesterol levels are the main modifiable risk factor for CAD (2,5,6).
Dyslipidemias are multifactorial traits and can be influenced by the environment, such as life habits and by genetic factors.Some studies in other populations have demonstrated that many genes can influence lipid levels, as the APOB and APOE genes (1,7,8,9).
Given the importance of dyslipidemias as a public health problem in Brazil, the aim of this study was to investigate if the R3500Q of APOB gene and ε2, ε3 and ε4 alleles of APOE gene influence the lipid profile in a Southern Brazilian cohort.

Statistical Analyses
To proceed with the statistical analysis, we classified the samples into groups above and below the median for the variables TC, LDL-C, HDL-C and TG.Each variable, stratified in two groups by its median, was plotted as a dependent variable in a logistic regression analysis.χ2 tests were performed using Clump (18) to test for Hardy-Weinberg equilibrium, and to compare allele proportions between groups above and below the median.Furthermore, the G 2 -test was used to compare genotype proportions between groups.The probability value for the comparative tests were considered significant at p < 0.05 (5%).

Polymorphisms of APOE and APOB genes and dyslipidemias in a South Brazilian Cohort
Gasparin et.al.

RESULTS
Table 1 shows the descriptive statistics for the variables analyzed in the present study.
Regarding the R3500Q mutation of APOB gene, we observed that the genotype TC levels in the sample stratified by APOB gene R3500Q site genotypes are shown in Table 2. Allele frequencies between groups were significantly different, with the group above the median for TC having a higher frequency of the A allele (p = 0.029).Comparisons between carriers of the rarer allele (AA and GA) and homozygotes for the common allele (GG) revealed a tendency to a higher frequency of carriers of the rarer allele in the group above the median for the TC (p = 0.053).Logistic Regression Analysis, using TC stratified as below and above the median as a dependent variable, and R3500Q site genotypes (in the tested dominant model GG and GA are dominant over AA), gender and age as independent variables, identified the A allele of APOB gene as an independent risk factor to increased TC levels (± 10377.78β = 21.326,p = 0.001).

DISCUSSION
The purpose of the present study was to investigate if there is an association between the R3500Q (APOB gene) mutation and variants of APOE gene with lipid levels in a sample of a Southern Brazilian population of self-declared European ancestry.In our work the comparisons between individuals stratified by lipid profile markers levels, revealed significant differences in allele frequencies of genes involved in lipid metabolism, and thus possibly contributing to the normal variation in the lipid levels of this population.
Regarding the analyses of APOB gene, it was observed that the frequency of the rarer allele (A) was significantly higher in individuals with TC above the median and from the multiple logistic regression analysis, it was found that the R3500Q mutation is a risk factor, independent of age and gender, for increasing TC levels.The independence among the factors analyzed is very relevant, because the lipid levels are influenced by environmental, genetic factors and by gender, thus these factors could influence metabolic pathways and thus alter the cholesterol concentrations (6,19,20,21).Our results are in agreement with the postulated effect of R3500Q mutation, which interferes with the conformation of the APOB 100 binding domain with the B / E receptor and thus reducing LDL-C affinity and consequently increasing serum levels of LDL-C and TC (12,22,23).Furthermore the R3500Q mutation is responsible for familial defective apolipoprotein B-100 (FDB) and its influence over lipid profile is studied worldwide (7,24,25).Therefore, our research contributes with data for a better understanding of genetic variants that influence changes on lipid levels in a Brazilian population sample.
According to a genome-wide association study (GWAS) there is a cluster of single nucleotide polymorphisms in the region of the APOB gene which is strongly associated with LDL-C levels (7).Furthermore they observed that the R3500Q mutation is strongly associated with LDL-C (7).Similarly to our study, Shen and cols.(7) also found an influence of this mutation over lipid profile and Nelken and cols.(8) observed the influence of this polymorphism on augmenting cholesterol levels in children and adolescents and thus suggesting that this genetic factor influences serum lipids levels early in life (7,8).
The effect of the APOE polymorphisms in serum lipids levels varies in different populations and represents from 4 to 15% of LDL-C levels variation (26).In different populations the ε2 allele was associated with low levels of LDL-C, whereas the ε4 was associated to high levels of LDL-C (1,9).We observed that the ε4 allele was more frequent in subjects with higher levels of TC and LDL-C, while the ε2 allele is more frequent in subjects with higher concentrations of HDL-C, forming a protective effect, since one function of those lipoproteins is removing the excess of LDL-C from the circulation (19).
Considering the ε4 allele influence over dyslipidemias and atherogenity its analyses is very important, specially because the consequences resulting from high lipid levels overwhelm the public health system.The atherogenity of the ε4 allele is probably due to its high affinity for apoE-binding receptors, leading to increased hepatic cholesterol pool which has the potential to down-regulate the LDLR, resulting in an accumulation of LDL-C and in an increased risk of atherosclerosis (27).ApoE3 and apoE4 bind to the LDLR with similarly high affinity, but the binding of apoE2 is somehow two orders of magnitude weaker (28).

Participants
Polymorphisms of APOE and APOB genes and dyslipidemias in a South Brazilian CohortGasparin et.al.BrazilianJournal of Implantology and Health Sciences Volume 6, Issue 1 (2024), Page 992-1012.The study sample consisted of 214 apparently healthy adults (41.90 years ± 9.7 years), 30 men and 184 women.Considering that the study participants constituted a population sample, no specific pathology was used as an inclusion criteria.All individuals in the sample were Euro-Brazilian residents in the city of Curitiba-PR (Southern Brazil).This project was approved by the Institutional Ethics Committee and informed consent was signed by all participants.Lipidogram Blood samples were collected in the morning after 12 hours of fasting, to perform measurements of total cholesterol (TC), high density lipoprotein (HDL-C), low density lipoprotein (LDL-C) and triglycerides (TG) by standard automated methods.DNA analysis DNA was extracted from peripheral blood samples by a salting-out method (17) and diluted to a final concentration of 20 ng/µL.R3500Q mutation (rs5742904) of APOB gene and the alleles ε2, ε3 and ε4 of APOE gene (rs7412 -and rs429358 ) were identified by TaqMan SNP Genotyping Assay (Applied Biosystems).Each reaction had 3.0µL of Master Mix, 1.7 µL of MiliQ Water, 0.3 of µL of primer and 3.0 µL of DNA and the reactions were performed in Mastercycler realplex 2 according to the following steps: (1) 50°C / 2 min, (2) 95°C / 10 min, (3) repeated 50 times 95°C / 15 sec interspersed with 62ºC / min.
Age is expressed in years.HDL-C, LDL-C, TG and TC are expressed in mg/dL.HDL-C: high-density lipoprotein cholesterol; LDL-C: low-density lipoprotein cholesterol; TG: triglycerides; TC: total cholesterol.Polymorphisms of APOE and APOB genes and dyslipidemias in a South Brazilian CohortGasparin et.al.BrazilianJournal of Implantology and Health Sciences Volume 6, Issue 1 (2024), Page 992-1012.The distribution of allele and genotype frequencies of the APOE gene are shown in Table 3, for the whole sample and for the sample stratified as above and below the median of TC, LDL-C, HDL-C and TG.By analyzing the data in the groups above and below the median, significantly higher frequencies of the ε4 allele were observed in the stratums above the medians of TC (X² = 5.29, p=0.021) and LDL-C (X² = 9.36, p = 0.0022).Furthermore, the ε2 allele was significantly more frequent in the group above the median of HDL-C (X² = 4.10, p = 0.0429).Regarding the genotypes, it was observed that ε3ε4 combination was more frequent in the group above the TC median (test-G = 17.33 and p = 0.0039); ε2ε4 had the highest frequency in the groups above the medians of LDL-C (Test-G = 18.09, p = 0.0028) and HDL-C (Test G = 17.08, p = 0.0044).

Table 1 .
Descriptive statistics for age and lipid profile of 214 individuals analyzed in this study.

Table 2 .
Distribution of allele and genotype frequencies of the R3500Q mutation of APOB gene in the groups above and below the median for

Table 3 .
Distribution of allele and genotype frequencies of the APOE gene in the groups above and below the median for the variables TC, LDL-C, TG and HDL-C

of APOE and APOB genes and dyslipidemias in a South Brazilian Cohort Gasparin
et. al.