Analysis of the Association Between Apolipoprotein E Polymorphism and Cardiovascular Risk Factors in an Elderly Population with Longevity

Helena Augustin Schwanke, Ivana Beatrice Mnica da Cruz, Ney Furhmann Leal, Rosane Scheibe, Yukio Moriguchi, Emlio Hideyuki Moriguchi

Objective

To establish the allelic and genotypic frequencies related to apolipoprotein E (ApoE) polymorphism and association of the genotypes with risk factors and cardiovascular morbidity in an elderly population with longevity.

An increasing number of genetic polymorphisms have been associated with the susceptibility to cardiovascular diseases, such as the apolipoprotein E (ApoE) gene. Studies have shown that the E4 allelic variant is associated with the mechanisms of development of atherosclerosis, dementia, and more recently, with the risk of bone fractures [1-5].

Methods

We analyzed 70 elderly patients aged 80 years or more who were part of the Projeto Veranpolis. We used the gene amplification technique through the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and cleavage with the restriction enzyme Hha I to identify the ApoE genotypes. The most frequent genotypes were compared considering biological variables and cardiovascular risks and morbidity.

The first genetic studies in cardiology were those of the association between ApoE polymorphism and coronary artery disease [6]. Apolipoprotein E participates in the metabolism of lipoproteins and plays a significant role in the transport of the cholesterol molecule [1 - ligand of the LDL receptor; 2 - activator of enzymes like hepatic lipase; 3 - hepatic production of very low density lipoprotein (VLDL)] [7,8]. In addition, this protein is related to immunoregulation and neurobiological routes (neuronal repair, remodeling, and protection). Apolipoprotein E is mainly synthesized in the liver (> 90%), but also in other tissues, such as the bowel, brain, lungs, kidneys, and macrophages, and secreted as a glycosylated protein. Apolipoprotein E polymorphism is located in the long arm of chromosome 19. Point changes are the molecular features of this polymorphism in the nitrogen bases, causing the replacement of 1 amino acid in the 112 and 158 positions of the peptide chain. The 3 most common isoforms are as follows: E2 (cysteine/cysteine), E3 (cysteine/arginine), and E4 (arginine/arginine). Of the 3 isoforms, E3 is the most frequently reported in the literature in the general population and E4 is rare in the centenarian population. The allelic frequency of ApoE varies in populations because the polymorphism of its locus ranges from 16% to 53% according to the population. Therefore, different phenotypes of ApoE exist, and they result from 6 genotypes as follows: 3 homozygous (E2E2, E3E3, and E4E4), and 3 heterozygous (E2E3, E3E4, and E2E4). Apolipoprotein E polymorphism changes protein both in its structure and function. The E2 isoform generates a protein whose binding to the LDL receptor is defective (in the rare situation of homozygosis and in the presence of other factors, such as diabetes mellitus, it may generate type III Fredrickson dyslipidemia or dysbetalipoproteinemia). On the other hand, the E4 isoform generates a protein whose affinity for the LDL receptor is 4 times greater than that for LDL itself [1-5,9]. Population studies have shown that the serum levels of LDL-cholesterol (LDL-c) and apolipoprotein B (ApoB) are higher in individuals with the E4 allele, intermediate in individuals with the E3 allele, and lower in individuals with the E2 allele [10,11].

A few studies have also associated the presence of the E2 allele with greater longevity because a higher frequency of that allele is observed in nonagenarians and centenarians [12,13]. These results, however, have not been reproduced in all the populations studied, and they seem not to follow a universal pattern. Excluding the possible methodological problems associated with this type of study reviewed by Marian [5] and Mansur [6], one of the hypotheses to be considered is that in chronic and degenerative diseases with a multifactorial origin, the set of genetic variations of a population associated with the social and environmental surroundings of the same population and with the processes related to biogerontology (biology of aging) canprocessed text:

A molecular weight marker of 10-bp ladder was used to analyze and identify the characteristic bands of each allele. Lipid values (mg/dL) for adults (>20 years) are as follows: Desirable (<200 TC), Borderline (200-239 TC), and Alterated (240 TC); Desirable (<130 LDL-c), Borderline (130-159 LDL-c), and Alterated (160 LDL-c); HDL-c (>35); and Desirable (<200 TG) and Alterated (200 TG). For biochemical assessment, venous blood samples were collected from study participants after a 12-hour fast, using the venoclysis system with the disposable vacuum device (Vacutainer). Tubes with no anticoagulant were used to quantify the lipid profile, Lp(a), glucose, and uric acid. Tubes with sodium citrate were used for fibrinogen quantification. The blood samples were centrifuged for 15 minutes in a Kubota KC-70 centrifuge at a velocity of 3,000 rpm to separate plasma from the remaining cells. Plasma was divided into five 1.0-mL aliquots in Eppendorf tubes with micropipettes. The plasma aliquots were refrigerated between -8 and 4°C for a maximum of 24 hours.