Lower Prevalence of Epsilon 4 Allele of Apolipoprotein E Gene in Healthy, Longer-Lived Individuals of Hellenic Origin

1 2 1 1 1 Nikolaos Stakias, Panagiotis Liakos, Efthimia Tsiapali, Maria Goutou, and Georgios N. Koukoulis

1Department of Internal Medicine, University Hospital, and 2Laboratory of School of Medicine, University of Thessaly, Larissa, Greece.

Apolipoprotein E (ApoE), and especially its e4 isoform, is considered a risk factor predisposing to coronary heart disease. We hypothesized that the absence of e4 allele offers a better chance for longer life. So we compared the prevalence of ApoE genotypes in 80 healthy aged individuals (HAI) (.80 years) and 391 Greek adults (median age 43 years) with ApoE genotype distribution consistent with the Hardy–Weinberg equilibrium (v2¼5.93, p . .05). ApoE genotypes were comparable in both groups with the exception of E3/3 and E3/4, which were significantly higher (87.50% vs 75.99%, p¼ .025) and lower (5.00% vs 13.19%, p ¼ .036), respectively, in HAI. The e2 and e3 allele frequencies were not different between the groups. The e4 allele was significantly less frequent in HAI compared to controls (3.1% vs 8.58%, p¼.020). Our results indicate an unfavorable effect of e4 allele on longevity that may be attenuated by environmental and/or other genetic factors.

frequency of e4 allele in healthy aged individuals would be expected to be lower compared to its distribution in the general population due to anticipated increased prevalence of CHD at an earlier age. This hypothesis was assessed in our study by examining the frequency of ApoE genotypes and alleles in a sample of healthy aged individuals and comparing it with the distribution of ApoE genotypes and alleles in a randomly selected population based sample of Greek adults.

E (ApoE) (for protein) is a poly- morphic glycoprotein that mediates the binding of lipid particles to specific lipoprotein receptors. A 4-exon gene located on the long arm of chromosome 19 encodes ApoE. Three major isoforms of ApoE—E2, E3, and E4—result from different amino acid substitutions at positions 112 and 158 in the 299-amino-acid protein. ApoE isoforms are encoded from separate alleles denoted, respectively, as e2, e3, and e4, which form three homozygous genotypes (E4/4, E3/3, and E2/2) and three heterozygous genotypes (E4/3, E4/2, and E3/2) (1,2). ApoE is synthesized predominantly in the liver but is also expressed in significant amounts in the brain, being important for membrane maintenance and re- pair (3). ApoE has been one of the most thoroughly studied genetic polymorphisms, particularly for its effects on lipid profile and coronary heart disease (CHD) risk. In compar- isons made to determine risk, the homozygous E3/3 geno- type is used as the referent.

M ETHODS

Participants

For the study of distribution of APOE genotypes and alleles in the population, a random sample of healthy adults was enlisted from a survey that was conducted in the region of Thessaly from 2001 through 2003. The goal of the survey was to gain insights into the prevalence of obesity and its metabolic consequences in adult Greeks. This group consisted of 391 individuals (194 men and 197 women; median age 43 years, range 19–60 years) and was used as a control group. Enrolling in the control group individuals younger than 60 years, we avoided a bias on the normal distribution frequency of ApoE genotypes due to increased CHD incidence and mortality rates in the Mediterranean area after the age of 60 years (12). A second group of healthy individuals older than 80 years was also recruited from the same region. This group consisted of 80 healthy persons (38 men and 42 women; median age 84 years, range 80–95 years) who were physically and mentally active. Individuals older than 80 years with diabetes mellitus (persons with fasting blood glucose . 7.0 mmol/L [125 mg/ dL], known cardiovascular disease, chronic renal failure, liver disease, or dementia were excluded from the study). Hypertension regulated satisfactorily with antihypertensive

Cardiovascular disease is today the main cause of both premature death and incapacity. It is generally accepted that the main risk factors for atherosclerosis, such as dyslipid- emia, hypertension, and diabetes mellitus, are at least partially gene-controlled (4). Many studies have indicated that genes coding for different plasma apolipoproteins, including the allelic variants of the APOE gene, may be associated with an increased risk for cardiovascular disease (5–8). ApoE polymorphism has been considered a risk factor for predisposition to CHD. Although older studies had shown apparently conflicting results (9,10), a recent meta-analysis of 46 studies demonstrated that e4 allele is a significant risk factor for CHD, whereas e2 allele has no effect (11). The increased risk for CHD is attributed to higher total serum cholesterol and low-density lipoprotein cholesterol levels (2).

Recent evidence indicates that the absence of e4 allele seems to be a favorable survival factor. If this is the case, the

Table 2. Relative Frequencies of Apolipoprotein E (ApoE) Alleles in the Control Group and the Healthy Aged Individuals

Table 1. Relative Frequencies of Apolipoprotein E (ApoE) Genotypes in the Control Group and the Healthy Aged Individuals

Control Healthy Aged Group Individuals Characteristics (n ¼ 391) (n ¼ 80) Age Distribution 19–60 y 80–95 y Fisher’s Exact Test ApoE Genotypes N (%) N (%) p Value E 2/2 2 0.53 2 2.50 .143 E 2/3 30 7.92 3 3.75 .238 E 3/3 288 75.99 70 87.50 .025 E 2/4 3 0.79 1 1.25 .536 E 3/4 50 13.19 4 5.00 .036 E 4/4 6 1.58 0 0 .596

Control Group Healthy Aged Fisher’s (n ¼ 391) Individuals (n ¼ 80) Exact Test ApoE Alleles (%) (%) p Value e2 4.88 5.00 1.000 e3 86.54 91.90 .066 e4 8.58 3.10 .020

Statistical Analysis