Hyperlipoproteinemia type III

ANNA KOWALSKA,1 IING:RID WIECHMANN,2 AND HUBERT WALTER2

Abstract Apolipoprotein E (apoE, protein; APOE, gene) is a component of very low density lipoprotein and high-density lipoprotein and plays an important role in lipoprotein metabolism. There are three common alleles of APOE (*2, *3, and *4), which encode the E2, E3, and E4 isoforms of the protein. Distribution of apoE isoforms shows marked variation among various ethnic groups. Direct phenotyping of human APOE in plasma was used to estimate APOE allele frequencies in 137 unrelated blood donors from 3 regions of Poland. The relative frequencies observed for the APOE*2, APOE*3, and APOE*4 alleles were 0.055, 0.839, and 0.106, respectively. The data have been compared with data found in other population groups. The frequency of the APOE*2 allele in Poles was among the lowest in Europe.

Apolipoprotein E (apoE, protein; APOE, gene), a 34-kDa component of the plasma lipoprotein system, is involved in cellular uptake of specific lipoproteins. The polymorphic nature of APOE was established using isoelectric focusing (Utermann et al. 1977). The three common apoE isoforms differ from each other by cysteine or arginine residues at positions 112 and 158: E2 (Cys112, 158), E3 (Cys112, Arg158), and E4 (Arg112, 158). The amino acid substitutions cause functional differences in binding affinity to apoE receptors among isoforms (Mahley 1988).

The genetic variability of APOE is associated with various human pathological conditions. Several studies have shown that the APOE*4 allele is a significant risk factor in cardiovascular disease and non-insulin-dependent diabetes mellitus (Davignon et al. 1988; Crews et al. 1991). Subjects carrying the APOE*4 allele have higher levels of total and low-density lipoprotein (LDL) cholesterol. A strong association between the APOE*4 allele and Alzheimer's disease has been reported recently (Corder et al. 1993). Numerous studies have demonstrated an increased frequency of the APOE*4 allele in familial and sporadic forms of Alzheimer's disease [e.g., Kamboh (1995)]. The E4 isoform shows high avidity binding to beta-amyloid peptide, possibly accelerating beta-amyloid deposition and senile plaque formation in brains of Alzheimer-affected individuals (Strittmatter et al. 1993).

Individuals with at least one APOE*2 allele tend to have lower levels of total and LDL cholesterol concentration than subjects homozygous for the APOE*3 allele. The APOE*2 allele seems to play a protective role in atherosclerosis and to promote human longevity. Its frequency is significantly increased among centenarians (Schachter et al. 1994). It has been suggested previously that people with the APOE*4 allele die earlier than people without it (Cauley et al. 1993). A small fraction of homozygotes for APOE*2 express type III and type IV hyperlipoproteinemia (Utermann et al. 1982).

APOE polymorphisms have been studied in various populations. However, no reports have documented APOE variability in Polish populations. The disease patterns associated with APOE polymorphism in different regions of Poland also remain unknown. The aim of the present study was to estimate frequencies of apoE isoforms in the general Polish population. The analysis is a first step to further studies on the association between APOE phenotypes and genotypes and cardiovascular disease, Alzheimer's disease, and vascular dementia-aimed toward understanding the role and effect of APOE on the development of multifactorial diseases in Poland.

Materials and Methods

In our preliminary study 137 serum samples from healthy Polish blood donors from three regions [Bialystok (n = 55) in eastern Poland; Poznan (n = 47) and Wroclaw (n = 35) in western Poland] (Figure 1) were collected and stored at -20 deg C until analysis.

Direct phenotyping of human APOE in plasma was performed using isoelectric focusing (IEF) and immunoblotting, according to the methods of Bailleul et al. (1993). Sera were treated with neuraminidase (type X, Sigma) and Tween-20 at 37 deg C for 2 hr. IEF in 1% agarose gel with 4 M urea (pH 47), prepared by mixing Ampholine (pH 5-7) and Pharmalyte (pH 4-6.5) (Pharmacia, LKB), was carried out at 12oC using a horizontal Multiphor electrophoresis unit (Pharmacia, LKB). The samples were prefocused for 1 hr at 3 W and 500 V and focused for 2 hr at 8 W and 1000 V. Afterward, IEF proteins were transferred onto a nitrocellulose membrane. The blot was incubated with a solution of a mouse anti-human APOE (50 ug/ml, Boehringer) in PBS for 2 hr, followed by washing in PBS with 0.1% Tween-20 and a second incubation with the antibody antimouse IgG-POD conjugated with peroxidase (Boehringer). The final protein pattern was visualized by blot staining with a substrate solution composed of 0.1 g of diaminobenzidine and 45 (mu)l H^sub 2^O^sub 2^ in 100 ml of PBS.

Hardy-Weinberg equilibrium and chi-square tests were used for statistical analysis.

Results and Discussion

Many techniques have been developed for APOE phenotyping. Most of them require purification of APOE by centrifugation (Zannis and Breslow 1982) or plasma delipidation (Menzel and Utermann 1986), serum treatment with guanidine-HCI (Havekes et al. 1987), or plasma microdialysis (Kamboh et al. 1988). In this study we used a simple specific agarose IEF immunoblotting method for screening APOE polymorphisms directly in plasma without prior treatments (Bailleul et al. 1993).

The distribution of APOE phenotypes and allele frequencies in 137 blood donors from 3 regions of Poland is summarized in Table 1. The five following APOE phenotypes were identified: APOE 2,3 (8%), APOE 2,4 (3%), APOE 3,3 (73%), APOE 3,4 (14%), and APOE 4,4 (2.2%). There was good agreement between the observed and the expected values according to Hardy-Weinberg equilibrium. No statistically significant differences in APOE allele distributions were found between the eastern and the western parts of Poland. Therefore we treated the three analyzed regions as one population. In our previous extensive studies on alpha-l-antitrypsin (PI) polymorphism we also found no significantly marked regional variation in PI allele distribution in five different parts of Poland (Kowalska et al. 1995). Based on this observation, we concluded then that the Polish population had been mixed strongly as a result of numerous migrations in the past, especially before and after World War II.

The frequencies of the most common APOE phenotypes (i.e., APOE 3,3, APOE 3,4, and APOE 2,3) in Poland and in other populations from Europe, the United States, China, and Japan are compared in Table 2. The Poles have one of the highest APOE 3,3 (73%) and the second lowest APOE 2,3 (8%) relative frequencies around the world. A higher frequency of the APOE 3,3 phenotype was observed in Greek Cypriots (76.42%), and a lower frequency of the APOE 2,3 phenotype occurred only in Hungarians (7.9%). In some Japanese and Chinese populations the APOE 2,3 phenotype was found more frequently than the APOE 3,4 phenotype.

The allele frequencies of the Polish sample were compared with frequencies found in other European countries (Table 3). There are regional differences in the distribution of APOE alleles among Europeans. The APOE*2 allele frequency in the Polish population is one of the lowest in Europe along with the Finnish and Sardinian populations. The frequencies of the APOE*4 allele are higher in northern Europe (Sweden, 0.206; Finland, 0.231) than in southern Europe (Italy, 0.083; Sardinia, 0.052; Greek Cypriots, 0.07). The opposite trend occurs for the APOE*3 allele. There is strong correlation between increased APOE*4 allele frequency and disease incidence.

The E4 isoform is more effective than the E3 isoform in binding and clearance by the LDL receptor. This raises the intracellular cholesterol concentration in hepatocytes and reduces LDL receptor activity, leading to increased concentration of cholesterol and triglycerides in individuals with the APOE*4 allele. The high frequency of the APOE*4 allele in the Finnish population might explain why they have one of the highest incidence of coronary artery disease in the world (Ehnholm et al. 1986; Lehtimaki et al. 1990), whereas the low frequency of APOE*4 among the Chinese and Japanese could partially explain the lower incidence of cardiovascular disease in these populations with respect to Western countries (Hallman et al. 1991). Intermediate values of APOE*4 and APOE*3 allele frequencies in Poland support the existence of a regular north-south cline in their distribution in Europe.

Received 10 October 1997; revision received 23 January 1998.

1 Institute of Human Genetics, Polish Academy of Sciences, 32 Strzeszynska, 60-479 Poznan, Poland.

2 Department of Human Biology, University of Bremen, Bremen, Bremen, Germany.

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