Hyperlipoproteinemia type III

* Background-The apolipoprotein E alleles epsilon2 and epsilon4 have been reported as independent risk factors for coronary artery disease (CAD) and as predictors for the development of atherosclerosis.

Methods and Results.-We determined by polymerase chain reaction the distribution of apolipoprotein E polymorphism in 320 Saudi blood donors (1313), 96 CAD patients, and 40 control subjects who had undergone angiography. Compared to controls, only epsilon4 was elevated in CAD patients. More than 61 % (P

had angina, and 52.1 % (P

Conclusions.-Accordingly, epsilon4 may be associated with increased risk of CAD, whereas epsilon2 appears to be a predictor of several risk factors for atherosclerosis.

(Arch Pathol Lab Med. 1999;123:1241-1245)

Apolipoprotein E (apo, E) forms an essential component of the clearance medianism of lipoprotein remnants by serving as a ligand for low-density lipoprotein (LDL) and the apo E receptors.1 3 Apolipoprotein E is genetically polymorphic and has 3 codominant alleles (apo epsilon2, epsilon3, and epsilon4) at the apo E gene locus on chromosome 19, giving rise to 6 genotypes, 3 of which are homozygous (epsilon2/2, epsilon3/3, and epsilon4/4) and 3 heterozygous (epsilon2/3, epsilon2/4, and epsilon3/4).4,5 These apo E isoforms derive from nucleotide substitutions (cysteine to arginine) in codons 112 and 158, leading to differences in the affinity of the resulting individual phenotypes for LDL and apo E receptors, the LDL receptor activity, the apo E distribution among lipoproteins, the LDL formation rate, and cholesterol absorption.6,7 The ensuing alterations in triglyceride, cholesterol, and LDL metabolism may in turn enhance conditions for the development of atherosclerosis.8,9 The allele epsilon2 has been associated with lower levels of serum total cholesterol, LDL cholesterol, and apolipoprotein B compared to the allele epsilon3, whereas epsilon4 has been associated with higher levels. 10-13 In some populations, homozygosity for apolipoprotein epsilon2 has been shown to be the major genetic prerequisite for the familial lipoprotein disorder type III hyperlipoproteinemia .10,11,14-16 Individuals with the disease appear to be strongly predisposed to developing premature and accelerated atherosclerosis.17-20 The allele epsilon4 has also been associated with type IV hyperlipoproteinemia,21,22 hypercholesterolemia (type Ha and Ilb hyperlipoproteinemia),126 and raised blood cholesterol in normolipidemia,7,27 suggesting that both epsilon2 and epsilon4 are reliable indicators for the development of atherosclerosis.19,28

Because of the association of the apo E alleles with various atherogenic factors, both alleles have been suggested to be risk factors for coronary artery disease (CAD).29 However, evidence for an association of the apo E gene polymorphism and cardiovascular diseases is not consistent. To begin with, while some investigators found a higher frequency of the allele epsilon4 in survivors of CAD,30 others have reported no significant difference 12 or even a lower frequency in these patients.31 A lower frequency of the apolipoprotein epsilon2 /3 phenotype has been observed in patients with angiographically documented coronary heart disease (CHD).32 Other studies have also shown heterogeneity of the apo E phenotype frequency among populations of different regions, ethnic populations, or geographically seperated groups within a particular population. Thus, some Asian populations appear to have reduced frequencies of both epsilon2 and epsilon4,33-37 whereas Northern European populations exhibit higher frequencies of the allele epsilon4. 38,39 It appears, therefore, tht the importance of the apo E polymorphism varies depending on ehnicity and the prevailing regional environmental parameters for each individual population.

In the present study, we determined the distribution of the apo E genotypes in the Saudi male population and evaluated its potential relevance to CHD as a whole. We further examined the possible relevance of the association of some important classic risk factors for CHD with the apo E polymorphism as predictors of the disease in this population.

METHODS

Study Population

The study population comprised 96 Saudi patients (83 men, 13 women) with angiographically documented coronary artery narrowing exceeding 75% (CAD group) who were admitted for coronary angioplasty at King Faisal Specialist Hospital and Research Centre, Riyadh, Saudi Arabia. Forty individuals (19 men, 21 women) who underwent angiography for surgical procedures unrelated to CHD were used as controls (CON group). Conventional risk factors for CHD, such as family history, hypertension, diabetes mellitus, hypercholesterolemia, and smoking, were determined through interviews with the patients or from a review of their medical records. Additionally, 320 healthy Saudi blood donors (317 men, 3 women) visiting the Blood Donor Clinic of the King Faisal Specialist Hospital and Research Centre (blood donor group) were recruited so we could evaluate the apo E genotype frequency in the general population. Informed written consent was obtained from all patients or family members prior to participation in the study. The study was performed in accordance with the rules and regulations established by the hospital's ethics committee.

Determination of Apolipoprotein Genotypes

Blood was obtained using standard methods in EDTA tubes, and genomic DNA was processed from 5 to 10 mL whole blood according to procedures described by Miller et al.40 The apo E genotypes were determined by polymerase chain reaction (PCR) in a DNA thermal cycler (Pharmacia, Uppsala, Sweden) using the oligonucleotide primers 5'-AGAATTCGCCCCGGCCTGGTACAC-3' (sense) and 5'-TAAGCTTGGCACGGCTGTCCAAGGA-3' (antisense). These primers were designed to encompass the polymorphic region of amino acids 112 and 158 of the apo E gene. A master PCR reagent mixture was prepared such that each 50 (mu)L contained 5 (mu)L of X 10 reaction buffer (Perkin-Elmer Corporation, Norwalk, Conn); 200 pmol/(mu)L each of deoxyadenine triphosphate, deoxyguanine triphosphate, deoxycytosine triphosphate, and deoxythymidine triphosphate in Tris HCl buffer; 5% dimethylsulfoxicle; 2.0 ng of each primer; 1 unit Taq DNA polymerase; and 10 ng genomic DNA. Samples were overlaid with light mineral oil, the mixture was denatured at 95 deg C for 5 minutes, and the PCR reaction was carried for 40 cycles under the following conditions: denaturing at 95 deg C for 45 seconds, annealing at 59 deg C for 45 seconds, and extension at 72 deg C for 2 minutes, with the final cycle extension running for 10 minutes. The 244base pair (bp) PCR product was digested overnight at 37 deg C with HhaI (Pharmacia, Uppsala, Sweden). The amplified products underwent electrophoresis on a 4% Metaphor gel (FMC Products, Rockland, Me) in TAE buffer containing 0.5 (mu)g/mL, ethidium bromide, and the amplicons were determined using bp marker pBR 322 DNA-Msp digest (New England BioLabs, Beverley, Mass). The Student 2-tailed t test for the comparison of sample groups, X^sup 2^ test for independence, and Fisher exact test or analysis of variance were performed to compare the differences between the groups when appropriate. A 2-tailed P value of less than .05 was considered to indicate an association between variables.

RESULTS

Three groups of individuals were included in this study. The mean age of the first group, which included 320 healthy Saudi blood donors, was 25.1 +/- 0.4 years (men, 25.0 +/- 0.4 years; women, 33.3 +/- 2.7 years). The predominance of the male population in this group (317 men, 3 women) was due to a lack of female blood donors visiting our Blood Donor Clinic at the time of the study. Therefore, the genotyping of this group provided information on the distribution of the apo E genotypes in the Saudi male population only. Baseline clinical data for the CON and CAD groups are given in Table 1. On admission, 61.5% (P

Digestion of the 244-bp amplicon produced 6 genotypes: epsilon2/2, epsilon3/3, epsilon4/4, epsilon2/3, epsilon2/4, and epsilon3/4. Homozygote epsilon2/2 resulted in 91-, 83-, 38-, and 19-bp fragments; epsilon3 / 3 in 91-, 48-, 38-, and 35-bp fragments; and epsilon4 / 4 in 72-, 48-, 38-, and 35-bp fragments. Heterozygote epsilon2/3 produced 91-, 83-, 48-, and 38-bp fragments; epsilon2/4, 91-, 83-, 72-, 48-, and 35-bp fragments; and epsilon3/4, 91-, 72-, 48-, 38-, and 35-bp fragments. A representative gel showing fragment patterns of these genotypes is shown in the Figure. The genotype epsilon3/3 and allele epsilon3 were significantly the most abundant, and epsilon2 was the least common allele in the 3 groups (Table 2). In the blood donor group, the genotype epsilon3 / 3 was found in 72.8%, while both epsilon2 / 2 and epsilon4 / 4 were each found in 1.3% of the members of this group, leading to a frequency of 84.4% for epsilon3, 3.6% for epsilon2, and 12.0% for epsilon4. In the CON group, epsilon3 / 3 was found in 77.5%, and none of the other main genotypes, including epsilon2/2 and epsilon4/4, were detected. The allele frequency in the CON group was 88.8% for epsilon3, 5.0% for epsilon2, and 6.2% for epsilon4. In the CAD patients, epsilon3/3 was identified in 70.8%, epsilon2/2 was identified in 1.0%, and epsilon4/4 was not present. Compared to the CON group, the allele epsilon4 was elevated in the 2 patient groups, while epsilon2 was reduced in the blood donor group and remained unchanged in the CAD group. These differences were not, however, statistically significant based on Fisher exact tests.

Only 1 patient carried the genotype epsilon2/2. This patient was a female CAD patient who presented with angina pectoris and who had a history of hypertension, diabetes mellitus, and elevated total cholesterol and serum triglyceride levels. Furthermore, 71.4% of all patients carrying the genotype epsilon2/3 presented with angina, and 57.1% had diabetes mellitus. As shown in Table 3, comparison of the relative distribution of the patient genotypes within the individual classic risk factor group suggested a strong association of the allele epsilon4 with hypertension, 3-vessel disease, and restenosis resulting from the genotype epsilon3/4.

COMMENT

In this study, we determined the distribution of the apo E genotypes in healthy blood donors to evaluate its relevance to CHD in the Saudi population. We also studied the relationship of the classic risk factors with apo E polymorphism and its implications for the prevalence of these disorders in this population. We established that epsilon3/3 is the most abundant genotype in the Saudi male population, and that epsilon2/2 and epsilon4/4 make about equal contributions. The low frequency Of epsilon2/2 and epsilon4/4 in the male population explains our failure to detect the epsilon4/4 genotype in any of the other 3 groups and our finding of only 1 patient carrying the genotype epsilon2 / 2. This genotype distribution follows a pattern similar to that observed in other populations in close proximity in the genetic lineage.1 Some studies have recently shown that the frequencies of both alleles epsilon2 and epsilon4 are lower in Asian than in Western populations,33 suggesting their selective association with CAD on a regional and ethnic basis. This might explain our observation of a relatively lower prevalence Of epsilon2 in all 3 study groups compared to Western populations. The present study could not, however, detect any significant differences among the 3 groups in the prevalence of the allele epsilon2. This is in contrast to previous findings that suggested epsilon2 is elevated in association with CAD.41 On the other hand, conflicting observations have been made regarding the role Of epsilon2 in the manifestation of these disorders.42 Studies in other populations have also been unable to demonstrate an association between increased risk of CAD and an elevation in epsilon2. 33,38 The overall findings of the present study suggest that neither of the 2 alleles predisposes individuals to CHD in the Saudi population. This conclusion does not quite conform to findings that have suggested an increased risk of CAD with an elevation in epsilon4. 43,44 Put together, these observations imply that the relevance of apo E polymorphism as a risk factor for coronary heart disorders varies from one population to another and must be established individually.

Arguments supporting an important role of apolipoprotein epsilon4 as a risk factor for CHD have been based mainly on observations of its influence on lipid levels in various populations .3,8,45 Compared to epsilon3, apolipoprotein epsilon4 has been associated with higher plasma cholesterol levels, whereas epsilon2 has been associated with lower levels.7,13-17 Some studies have suggested that epsilon2 constitutes a negative risk factor for atherosclerosis through its hypocholesterolemic effect,21 whereas others have proposed an atherogenic effect in some cases and an antiatherogenic effect in others.27

We were also interested in evaluating the association of apo E polymorphism with the established risk factors for CHD. Although we found no significant relationship between apo E genotyping and CHD, the abundance of the baseline clinical variables and the genotype distribution did show some interesting relationships. To begin with, the results clearly indicate that a report of angina pectoris on admission was a reliable predictor of CAD. In our study population, the only epsilon2 / 2 carrier was a female CAD patient exhibiting a combination of all risk factors, except myocardial infarction and family history. Furthermore, diabetes and elevated lipid levels appeared to be good indicators for CAD and the extent of vessel disease. The presence of the allele epsilon2 was not only associated with angina and diabetes mellitus, but also with important predictors of atherosclerosis, such as elevated serum levels of triglycerides and total cholesterol, whereas epsilon4 was more closely related to hypertension and 3-vessel disease. It appears, therefore, that apo E genotyping is more relevant for establishing the prevalence of certain risk factors predisposing individuals to atherosclerosis than as an indicator of CHD per se. Furthermore, individual risk factors are probably more reliable predictors for CAD than genotyping in this population.

Although family history and hypertension did not appear to be very important indicators for CAD in general, it was interesting to note that the female patients were the primary contributors to these factors; 69.2% of the female patients with CAD were hypertensive. These results are indicative of a selective association between the genotypes of the female patients and certain risk factors for CHD. Further studies using larger populations should greatly enhance our understanding of this issue.

In summary, our results show that the distribution of the apo E genotypes in the Saudi male population is similar to that of other Asian populations. Compared to the CON group, epsilon4 may be associated with an increased risk of CAD, whereas epsilon2 is not. On the other hand, epsilon2 is closely associated with classic predictors of atherosclerosis, such as elevated serum triglycerides and total cholesterol, as well as hypertension and diabetes.

We are thankful to Ella Williams for collection of patient data; to Gamal El-Din Mohamed, PhD), for his assistance in the statistical evaluation of the results; and to Chelton Jenkins, Hussein Farhoud, and the Blood Donor Clinic team of the King Faisal Specialist Hospital and Research Centre for their assistance in the procurement of blood samples for the blood donor group.

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Accepted for publication April 29, 1999.

From the Departments of Biological and Medical Research (Drs Dzimiri, Meyer, Hussain, and Ms Basco) and Cardiovascular Diseases (Drs Afrane and Halees), King Faisal Specialist Hospital and Research Centre, Riyadh, Saudi Arabia.

Reprints: Nduna Dzimiri, PhD, Biological and Medical Research Department (MBC-03), King Faisal Specialist Hospital and Research Centre, PO Box 3354, Riyadh 11211, Saudi Arabia.

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