Apolipoprotein E allele frequencies in an Italian population: Relation to age and lipid profile

G. Ruiu, M. Cassader, R. Gambino, N. Alemanno, F. Demichieli, A. Pagano, F. Veglia, and m n.
Istituto di Medicina Interna, Universita di Torino, Torino, Italy
Unita di Epidemiologia, Istituto Scientifico Ospedale S. Raffaele, Milano, Italy

Complications (10-13) have suggested that the frequency of allele 4 gradually decreases with age. Evidence of this change, coupled with an increase in 2, has been provided by a number of investigations (14-17).

ABSTRACT: Apo E phenotype and plasma Tg, Chol, LDL-Chol, HDL-Chol and Apo B levels were determined in a sample of 228 healthy Italian subjects (124 men and 104 women) aged 18-93. The allele frequencies were: 2=0.070; 3=0.829; 4=0.101 (among the lowest values in the literature). Division of the sample into four age groups indicated that allele 4 frequency decreased with age to zero in persons aged over 75. Covariance analysis of the influence of each allele on plasma lipids showed that allele 4 was significantly associated with the highest Chol, LDL-Chol and Apo B levels. These data are evidence of the influence of allele 4 on Chol metabolism in an Italian population and show that its frequency decreases with age.

This study provides evidence of the distribution of Apo E phenotypes and their relationship to plasma lipids and allele 4 frequency patterns in a male and female Italian population divided into four age groups.

MATERIALS AND METHODS
Subjects: Plasma samples for Apo E phenotyping were collected in the morning after an overnight fast from 124 male and 104 female subjects aged 18-93, divided into four age groups: under 65, 66-70, 71-75 and over 75. The population consisted of medical students, hospital staff members and persons over 60 who were able-bodied inmates of a rest-home or registered for analyses at the laboratory of the San Giovanni Hospital, Torino. Subjects with diabetes or liver, kidney or thyroid diseases were excluded.

INTRODUCTION: The 299-amino-acid glycoprotein apolipoprotein (Apo) E is primarily a component of chylomicra and VLDL (1) and acts as a ligand for the LDL receptor (Apo BI00-E receptor) and the remnants receptor (Apo E receptor) (2). Human Apo E displays a genetically-determined polymorphism (3,4); three alleles on the same locus encode for the three E2, E3 and E4 isoforms to produce three homozygous and three heterozygous phenotypes. Differences in the isoelectric points of the three isoforms allow phenotyping by isoelectrofocalization (IEF) (5,6).

Studies of allele distribution in several populations have revealed a lack of uniformity in allele 4 distribution (7). The major importance of this finding lies in the fact that the three alleles influence cholesterol (Chol) metabolism. Bearers of one or more allele 4 alleles display high total Chol and LDL-Chol levels, whereas the opposite is true for allele 2 (7-9). The observation of enhanced allele 4 frequency in patients with atherosclerotic cardiovascular complications suggested that its frequency gradually decreases with age, and evidence of this change, coupled with an increase in allele 2, has been provided by a number of investigations (14-17).

Preparation of samples: 1 mg/mL EDTA-Na2 plasma samples were centrifuged for 30 minutes at 2500 rpm at 4C in a J6B centrifuge (Beckman, Palo Alto, CA, U.S.A.), and stored at -20C until processed. Chol and triglycerides (Tg) were measured enzymatically. HDL-Chol was determined after precipitation of Apo B-containing lipoproteins with heparin and manganese chloride (18). LDL-Chol was calculated according to Friedewald et al. (19). Plasma Apo B was determined by the turbidimetric method (Poli Diagnostici, Milano, Italy).

Apo E phenotyping: Plasma samples (10 µL) were delipidated with acetone and ethanol (2.5 mL) 1:1 v/v for 4 hours at -20C, resuspended with acetone and ethanol (2.5 mL) for 1 hour, and washed in diethyl-ether (1 mL). The final precipitate was dried under nitrogenWith another Italian study (27), the Mantel-Haenszel X2 test showed an increase in 3 frequency with age (p=0.013), coupled with a decrease in 4 frequency (p=0.015) [Table 3], which was also more marked in males (data not shown). The average effect of the 2 allele was to decrease total Cholesterol, LDL-Cholesterol, and Apo B by 16.9, 18.5, and 7.3 mg/dL, respectively, in men and by 17.7, 14.5, and 12.9 mg/dL, respectively, in women. By contrast, the average effect of the 4 allele was to increase the previous lipid plasma levels by 9.3, 8.3, and 9.1 mg/dL, respectively, in men and by 10.5, 10.8, and 5.6 mg/dL, respectively, in women. The three alleles are an important factor in determining the plasma levels of the lipoproteins that transport cholesterol (9,28). In vitro and in vivo studies have identified the physiopathological mechanism responsible for the differences in allele behavior whereby 2 and 4 are respectively associated with the lowest and highest total Cholesterol, LDL-Cholesterol and Apo B levels (29). The average effect findings show that the values of these three parameters are reduced in 2 carriers and increased in 4 carriers. Covariance analysis confirmed the positive correlation between Cholesterol, LDL-Cholesterol and Apo B levels and 4 [Table 4]. This correlation had already been established in larger case studies (14-16) and is indicative of the impact of 4 on cholesterol metabolism.