ATHEROSCLEROSIS

Apolipoprotein E and B polymorphisms - longevity factors assessed in nonagenarians

UDepartmeni of Internal Medicine, ‘Biocenter Oulu. Universitv of Oulu, Kajaanintie 50. FIN-90220 Oulu. Finland cNational Public Health InsMule, Helsinki. Finland

(Received 6 July 1993; revision received 22 September 1993; accepted 24 September 1993)

Abstract

To test if the prevalence of genetic risk factors for coronary heart disease (CHD) is low in individuals who have reached an extremely old age, the allele frequencies of apolipoprotein E (apo E) and B (apo B) polymorphisms and plasma lipoprotein(a) levels were investigated in nonagenarians and in younger control groups. The frequency of the ~4 allele of apo E was significantly lower in the nonagenarians than in the middle-aged and young adults (P < 0.05). Also, the frequency of EcoRI allele R- of apo B was low in the nonagenarians, whereas the allele frequency for the J&I polymorphism of apo B and plasma lipoprotein(a) concentrations did not differ between the nonagenarians and the younger groups. These findings strongly suggest that the presence of these potential genetic risk factors for CHD, namely the ~4 allele of apo E and the R- allele of apo B, decreases the probability of an individual reaching an extreme-

Key words: Apolipoproteins; Cholesterol; Lipoprotein(a); Lipids; Arteriosclerosis; Aging; Genetics; Longevity

1, Introduction

tors are involved in determining an individual’s susceptibility to CHD [3]. In a population with a high incidence of CHD, a low frequency of genetic risk factors for CHD could contribute to longevity so that possible protective genotypes would be enriched in extremely old cohorts.

* Corresponding author, Tel.: 358-81-3154100; Fax: 358-81- 31 55423.

Apo E is one of the major protein constituents in very low density lipoprotein (VLDL) and plays an important role in lipoprotein metabolism [4,5]. Apo E has three common isoforms, namely E2, E3 and E4, which are coded by the alleles ~2, ~3 and

Part of this work was presented at the

th Scientific Sessions of the American Heart Association, Anaheim. CA, I I-

November

and as an abstract in Circulation.

(

) Suppl. II.

-

0021-9150/94/$07.00 0 1994 Elsevier Science Ireland Ltd. All rights reserved. SSDI 002 1-9150(93)05157-Z

~4 respectively at a single locus on chromosome 19. These alleles define six apo E phenotypes: E2/2, E2/3 and E2/4 (referred to as E2 category), E3/3 (E3 category), E4/3 and E4/4 (E4 category). The E4 category is associated with high serum total and low density lipoprotein (LDL) cholesterol levels in many populations [4-61. In Finland, the prevalence of e4 allele is higher than in most other countries [6].

The control group (n = 260) consisted of healthy volunteers who worked in the Oulu University Hospital (n = 200, age 21-62 years) [14], and of healthy employees of a telecommunications com- pany (n = 60, age 35-64 years) [ 151. The control subjects were divided into two age categories: 40 years or younger (n = 124) and over 40 years (n = 136). The groups are referred to as young adults and middle-aged adults respectively. One individual in the latter group was diabetic. None of the control subjects was on lipid lowering drug therapy.

Apo B is the sole protein constituent in LDL, the major cholesterol carrying lipoprotein. Re- striction fragment polymorphisms (RFLPs) in the apo B gene, detected with the enzymes X&I and EcoRI have been shown to be associated with the occurrence of CHD [7,8]. Several epidemiological studies have indicated that another apo B- containing lipoprotein, lipoprotein(a) (Lp(a)) is also atherogenic [9- 111. The genetically determin- ed polymorphism of apolipoprotein(a) (ape(a)) is related to plasma Lp(a) concentrations [12], so that ape(a) isoforms predict risk for CHD through their effects on Lp(a) concentrations in some populations [ 131.

2.2. Methods

A venous blood sample was obtained into ethylenediaminetetraacetic acid (EDTA) tubes after an overnight fast, and the plasma was separated by centrifugation at 1200 x g for 10 min (+4”(Z). LDL and high density lipoprotein (HDL) cholesterol were estimated from the fresh plasma as described in the Lipid Research Clinics Manual of Laboratory Operations [ 161.

The aim of the present study was to examine whether polymorphisms at the apo E and apo B gene loci and plasma Lp(a) levels are associated with longevity in a population with a high risk for CHD. Specifically, the following questions were addressed: (i) is the prevalence of various apo E phenotypes in nonagenarians different from that in younger age groups? (ii) are the allele frequen- cies for the EcoRI and XbaI RFLP polymorphisms of the apo B gene in nonagenarians different from that in other age groups? and (iii) do the plasma Lp(a) levels change with increasing age?

Cholesterol and triglycerides (enzymatic colori- metric methods, kits of Boehringer Diagnostica, catalogue nos. 236691 and 701912 respectively, Boehringer Diagnostica, Mannheim GmbH, FRG) were analysed using a Gilford IMPACT 400E Clinical Chemistry Analyzer (Gilford In- struments Laboratories Inc., Oberlin, OH).

The apo E phenotype was determined from the plasma with isoelectric focusing and immunoblot- ting techniques [6,17]. The first antibody was a rabbit antibody to human apo E. The second anti-

2. Subjects and methods

2.1. Subjects

characteristics of the study groups

All the subjects of 90 years or older who lived in the town of Oulu (total population 100 000 inhabitants) were identified from community records. Eighty-seven women and 8 men (Table 1) (70% of the cohort, median age 93 years) gave an informed consent and volunteered for the study that had been approved by the Ethical Committee of the Oulu University. Of the study group, 30% lived at home, the rest being at institutions for the