---

Genetic polymorphism of apolipoprotein E in peripheral arterial disease
(Portuguese PDF version)

Antônio Carlos Brandão,¹ Sidney Pinheiro Jr.,² Marcela Augusta Pinhel,³ Alexandre Maieiras Anacleto,⁴ José Maria Pereira de Godoy,⁵ Moacir Fernandes de Godoy,⁵ José Ernesto dos Santos,⁶ Dorotéia Rossi Silva Souza⁷

1. PhD, Associate professor, Department of Molecular Biology, School of Medicine of São José do Rio Preto (FAMERP), São José do Rio Preto, SP, Brazil.
2. Assistant professor, Department of Molecular Biology, FAMERP, São José do Rio Preto, SP, Brazil.
3. Trainee in Biology, Department of Molecular Biology, FAMERP, São José do Rio Preto, SP, Brazil.
4. Physician, Instituto de Medicina e Cardiologia de São José do Rio Preto, SP, Brazil.
5. Physician, Associate professor, Department of Cardiology and Cardiovascular Surgery, FAMERP, São José do Rio Preto, SP, Brazil.
6. Physician. Professor, Department of Medical Clinic, School of Medicine of Ribeirão Preto-USP, Ribeirão Preto, SP, Brazil.
7. Physician, Associate professor, Department of Molecular Biology, FAMERP, São José do Rio Preto, SP, Brazil.

Correspondence:
Antonio Carlos Brandão
Rua Garabed Karabashian, 411
CEP 15070-600 - São José do Rio Preto, SP, Brazil
E-mail: godoyjmp@riopreto.com.br

---

ABSTRACT

Objective: The objective of this study was to evaluate the genetic polymorphism of apolipoprotein E in peripheral arterial disease.

Method: A total of 61 Caucasian male patients aged between 38 and 79 years old were studied. All of them had clinical symptoms of arterial disease, later confirmed by angiography. A control group with 59 individuals was also included. Patients with renal disease, liver disease or diabetes mellitus were excluded from the study. The genomic DNA was extracted from 5 ml of peripheral blood leukocytes, collected in ethylenediaminetetraacetic acid (EDTA) tubes. The apolipoprotein E alleles were then investigated. Statistical analysis was performed using Fisher's exact test and odds ratio estimates.

Results: The ε3 allele was more frequent in patients than in control patients, whilst the 4 allele was more frequent in control patients than in the study group; even though, no significant difference was evidenced. Patients with occlusive peripheral arterial disease presented with a higher frequency of the ε3/ε4 genotype as compared to patients with aneurysmal peripheral arterial disease. No statistically significant differences were observed.

Conclusion: No evidence of an association between apolipoprotein E polymorphism and peripheral arterial disease was observed.

Key-words: genetic polymorphism, apolipoprotein E, atherosclerosis.

J Vasc Br 2004;3(4):317-22

---

Apolipoprotein E (apo E) is present on plasma lipoproteins, including high density lipoproteins (HDL), very low density lipoproteins (VLDL) and chylomicrons, as well as on products of lipolytic degradation, such as chylomicrons remnants and intermediate density lipoprotein (IDL). It was firstly identified in the 1970's¹ and hitherto it has been extensively studied specially because of its function in the lipid metabolism and its connection with the transportation of cholesterol out of the cell.

Apo E expression is recognized in different organs and glands of human beings, mice and rabbits.² However, substantial amounts of apo E mRNA are found in the liver, which is the primary source of this protein in plasma; apo E mRNA is also abundant in the brain, which releases apo E in the cerebrospinal fluid.³

Apo E is also synthesized by macrophages and it seems to play a significant role in the lipid metabolism and in the development of vascular diseases. It can bias the balance between cholesterol and smooth muscle cells of the arterial wall, affecting the progression or regression of atherosclerotic lesions. Besides controlling the platelet clustering and the lymphocytes proliferation, it also interacts with the extra cellular matrix, which affects the retention of lipoproteins in the arterial wall, the bio-feasibility of abducted citokynes and growing factors. It also controls the growing of smooth muscle cells.⁴

Apo E polymorphism was firstly referred by Utermann et al.⁵ Zannis et al.⁶ and Utermann et al.⁷ observed three major apo E common alleles: 2, 3 e 4. They code for isoforms E2, E3 and E4, which differ by the contents of cysteine and arginine at codons 112 and 158 in exon 4. Besides genetic variation, some modifications after translation may happen in the main apo E isoforms.²

The connection between apo E polymorphism and atherosclerosis was first described in a study carried out with patients with hyperlipoproteinemia type III with the apo E phenotype E2/2 and early coronary artery disease (CAD).⁸ One percent of the population is homozygous for apo E2, what leads to primary dysbetalipoproteinemia or type III hyperlipoproteinemia, when associated with other diseases as diabetes, hyperthyroidism or genetic dyslipidemia, with estimated frequency of 0.01 to 0.1% in this population.^2,9 Moreover, there are reports of an increased frequency of the 4 allele in patients with atherosclerotic peripheral arterial disease (PAD) and with aortic aneurysm.^10,11

An autopsy study performed during 10 years with men between 30 and 70 years-old evidenced that the 4 allele is associated with CAD only after the age of 53, thus suggesting that other factors or advanced age may be significant in the development of atherosclerosis.¹² Research on genetic polymorphism in humans have helped to clarify the apo E action mechanisms on cardiovascular disease. However, the global population lacks information on the identification and distribution of apo E alleles and their effect, especially in PAD.

The goal of the study we report on this article is to assess the association between apo E genetic polymorphism and occlusive or aneurysmal PAD in male Caucasian patients.

PATIENTS AND METHODS

Sixty-one male Caucasian patients, aged between 38 and 79 years old (mean and standard deviation = ± 8,6 years), with clinical signs and symptoms of PAD (claudication) and aneurysm were assessed in a random study. The diagnosis was confirmed by angiography and computerized tomography. Patients and control group were selected at random from ambulatories of the School of Medicine of São José do Rio Preto and the Instituto de Moléstia Cardiovascular de São José do Rio Preto, during 1 year (2001 to 2002). Individuals with renal or hepatic disease, diabetes mellitus, amputees and who had been submitted to previous treatment of atherosclerotic disease were excluded from the study and control groups. Among these, 39 presented atherosclerotic occlusion in the iliac, femoral and/or carotid arteries, and 22 had aneurysm of the thoracic, abdominal or thoracic abdominal aorta. The association between the occlusive disease and patients with aneurysm was not approached. The control group was composed of 59 male individuals, age range 43 to 80 years-old (mean and standard deviation = 59±9,2 years), with no clinical history of occlusive disease nor alterations in the clinical examination. All signed an informed consent form and the study was approved by the ethical committee of the Medical School of São José do Rio Preto (CEP-FAMERP).

Apo E polymorphism

The genomic DNA was extracted from 5ml of peripheral blood leucocytes collected in ethylenediaminetetraacetic acid (EDTA) tubes. The extraction was performed in three steps: 1) lysis of blood cells and denaturation by dodecyl trimethylammonium bromide (DTAB); 2) protein precipitation with chlorophorm; 3) DNA precipitation and resuspension with cetyltrimethylammonium bromide (CTAB).¹³

Polymorphic segments of the apo E gene were amplified by Polymerase Chain Reaction (PCR) with the primers P1:5´ -ACAGAATTCGCCCCGGCCTGGTACAC-3´and P2:5´-TAAGCTTGGCACGGCTGTCCAAGGA-3´, which complement the regions proximal to the polymorphic codons 112 and 158, located at the apo E exon 4.¹⁴ In the PCR we added: 0.5 µl of each deoxynucleotide (o.8 mM); 2.5 10X PCR buffer; 2,5 µl of dimethylsulfoxide 10%; 2,5 µl of each primer (2,5 mM); 0,2 µl of Taq polymerase (5 U/µl); 11 µl Milli Q water; and 2 µL of soluble genomic DNA (0,2 µg).

The initial DNA naturation was obtained at 94 oC for 5 minutes, the reaction was soon after subjected to 40 cycles of 94 oC for 30 seconds and 65 oC for 2 minutes, with a final cycle of 72 °C for 7 minutes. The result of the PCR was submitted to the restriction enzyme Hha I (5 U per reaction tube) in a water bath at 37 °C, overnight, for the cleavage of the amplified sequences of ε2, ε3 and ε4 alleles in specific loci (GCG) at codons 112 and 158, separating fragments with 91 base pairs (bp) and 83 bp (ε2), 91 and 48 bp (ε3), and 72 and 48 bp (ε 4). All six possible genotypes for apo E, ε2/ε2, ε2/ε3, ε2/ε4, ε3/ε3, ε3/ε4, ε4/ε4, were clearly discernible.

For the apo E analysis, DNA fragments were separated in a polyacrylamide gel electrophoresis 6% for about 3 hours, under a constant 200 V current, using TEB buffer (0.89M boric acid, 0.89M Tris, 0.025M sodium EDTA) dilute 10X.

The pBR 322 (Gibco) was used as DNA control, digested by the MspI restriction enzyme, yielding fragments up to 110, 90, 76 and 67 bp. After electrophoresis, the gel was stained with ethidium bromide (0.2 mg/l) for 5 minutes and DNA fragments were visualized under ultraviolet light, and photographed.

Statistic analysis

The allele and genotypic frequencies between patients and controls were compared with the Fisher's exact test. A similar procedure was carried out between patients with occlusive or aneurysmal PAD. The influence of alleles and genotypes in the disease was checked through the odds ratio, with confidence interval (CI) of 95%. An a error of up to 5% was accepted with significant P ≤ 0,05.

RESULTS

The distribution of alleles and genotypes of 61 patients and 59 controls is presented in Table 1. There is a higher presence of the ε3 allele in patients (0.893) than in the control group (0,814), followed by the ε4 allele with reduced prevalence in patients (0.074) than in the control group (0,135), although without significant difference (P = 0.2088). The same was true for the ε2 allele. The genotype ε3/ε3 was more frequent in patients (78,7%) than in the control group (69.5%), followed by the genotype ε3/ε4 (14.7 and 22%; P = 0.2994 e 0.3507, respectively). Genotypes with the ε2 allele were scarce in both groups.

Table 1 - Distribution of allele and genotypic frequencies of apolipoprotein E polymorphism determined by restriction enzyme Hha I analysis in patients with peripheral arterial disease and control group.

	Patient		Control		P
Allele	n	Frequency	n	Frequency
2	4	0.033	6	0.051	0.5343
3	109	0.893	96	0.814	0.099
4 Total	9 122	0.074 1.000	16 118	0.135 1.000	0.2088 --
Genotype	n	%	n	%
2/ 2	0	0	1	1.7	0.4917
2/ 3	4	6.6	1	1.7	0.3647
2/ 4	0	0	3	5.1	0.1158
3/ 3	48	78.7	41	69.5	0.2994
3/ 4	9	14.7	13	22	0.3507
4/ 4	0	0	0	0	--
Total	61	100.0	59	100.0	--

(--) = not applicable

 

Table 2 shows the distribution of alleles and genotypes for apo E in patients with occlusive and aneurysmal PAD. There are similarities between the two groups, with increased frequency of the ε3 allele (0.885 and 0.910, respectively), followed by the ε4 and ε2 alleles. The ε3/ε3 genotype was found in 81.8% of patients with aortic aneurysm and in 74.4% patients with occlusive PAD. The ε3/ε4 genotype was mostly present in patients with occlusive PAD (17.9%) as compared to those with aneurysmal PAD (9.1%), although without a statistically significant difference between the groups (P = 0.4674).

Table 2 - Distribution of allele and genotypic frequency of apolipoprotein E polymorphism determined by restriction enzyme Hha I analysis in patients with occlusive or aneurysmal peripheral arterial disease (PAD)

	occlusive DAP		aneurysmal DAP		P
Allele	n	Frequency	n	Frequency
2	2	0.025	2	0.045	0.6189
3	69	0.885	40	0.910	0.7682
4	7	0.090	2	0.045	0.4861
Total	78	1.000	44	1.000	--
Genotype	n	%	n	%	P
2/ 2	0	0	0	0	0
2/ 3	2	7.7	2	9.1	0.6147
3/ 3	30	74.4	18	81.8	0.7533
3/ 4	7	17.9	2	9.1	0.4674
4/ 4	0	0	0	0	0
Total	39	100	22	100	--

(--) = not applicable

Table 3 shows the odds ratio values for genotypes and alleles of apo E, comparing their association with PAD against the control group. Notice that all 95% CIs (Confidence Intervals) contain the unit, which is equivalent to the odds in groups. This fact indicates that the ε2, ε3 or ε4 allele and the genotype ε3/ε3 or ε3/ε4 do not influence apo E in PAD.

Table 3 - Odds ratio value for genotypes and alleles of apolipoprotein E (apo E) and the association with peripheral arterial disease (PAD)

Apo E polymorphism	Odds ratio - CI 95%
Allele
2	0.63 (0.17 - 2.30)
3	1.92 (0.92 - 4.02)
4	0.28 (0.24 - 1.31)
Genotype
2/ 2	0.32 (0.01 - 7.95)
3/ 3 2/ 4 2/ 3	1.62 (0.71 - 3.70) 0.13 (0.01 - 2.60) 4.07 (0.44 - 37.56)
3/ 4 4/ 4	0.61 (0.24 - 1.56) IC

CI = confidence interval; CI = impossible calculus.

DISCUSSION

The ε4 allele, sometimes mentioned as a risk factor for CAD and PAD in the literature,15,16 had low frequency in the present study especially in patients with PAD (0.074), although without statistically significant difference as compared to controls (0.135). The frequency in controls is reduced as compared to Caucasians. Prevalence varies from 0.140 to 0.227.16 The ε2 allele had low frequency in patients (0.033) and controls (0.051). These values were smaller than those found in the literature (0.072 to 0.130).16 The reduced frequency of the ε2 and ε4 alleles determined the high prevalence of the ε3 allele in patients (0.893) and controls (0.814). These numbers were higher than reported by other authors concerning Caucasian population, with a variation of 0.720 to 0.786.16

Although the polymorphic nature of the apo E gene is well known, its association with atherosclerotic cardiovascular diseases is polemic. A study with 77 men and 27 women with CAD showed significant increased prevalence of the ε4 allele in men (0.29) against women (0.15), although without statistical significance.15 On the other hand, another study carried out in Brazil with 50 women with CAD and mean age 48.9 showed a higher significant frequency of the 4 allele in patients as compared to controls (0.23 versus 0.11; P < 0.05). In this case, there was a significant prevalence of the genotype ε3/ε4 in patients (40%) than in the control group (14%).17

There are reports that confirm the association between the allele 4 and the slightly higher concentration of total cholesterol in these patients (227.3 mg/dl), when compared to the absence of this allele (206 mg/dl). In this case, there was a significant high risk of CAD in smoking individuals and carriers of allele ε4.18 Some studies show also an association between ε4 and an increase in the level of LDLc (low density lipoprotein cholesterol) in the early CAD, although there has not been established a relation between the disease and the apo E polymorphism.19

On the other hand, a study on ischemic vascular accident did not detect the relation between apo E polymorphism and the disease in a population over 75 years, with frequencies of 24% for the ε3/ε4 genotype in patients and 25% in the controls.12 The association of PAD and polymorphism of apo E is still obscure and polemic. The population of the referred study was 3,161 Americans of Japanese ancestry, age range 71 to 93, and they did not show association between PAD and diabetes, or smoking and apo E polymorphism.12 Although patients who entered our study were younger, they did not show an association between the presence of the ε4 allele and the PAD, regardless of the type of occlusive lesion: atherosclerotic or aneurysmal.

On the other hand, experimental studies carried out with handicapped animals for apo E confirmed an association with carotid, aortic, femoral and popliteal stenotic lesions.20 The apo E study in 260 healthy individuals detected a reduced frequency of the ε4 allele (0.100) as compared with the ε3 allele (0.827). However, other tests showed a significant incidence of occlusive carotid lesions in individuals who carried the ε4 allele as compared to those who carry the ε3 allele (P = 0.029).11 Besides, the presence of ε4 showed an association with atherosclerosis in non-diabetic individuals, thickening the carotid intimal layer, as compared to individuals of the genotype ε2/ε 3 or ε3/ε3.21 Nevertheless, a 2 to 4.5-year follow-up study with 57 patients with abdominal aortic aneurysm did not show an association between the allele ε4 and the lesion expansion.22

An anatomic-pathologic study involving 700 male individuals, aged between 33 and 70 years (mean = 53 years), made a relation between the frequency of the genotype ε3/ε4 and the expansion of atherosclerotic lesions in patients with coronary disease and abdominal and thoracic aorta disease, when compared to the genotype ε3/ε3.12 In this case, younger patients (below 53 years-old) had a significant P = 0.0085, while older patients had P = 0.041. The thoracic aorta presented a significant P = 0,014, while for the abdominal aorta it was not significant (P = 0,12), with no age influence. In the present study, patients had problems mainly in the femoral or abdominal arteries (occlusive PAD and aneurysmal PAD, respectively), and both lesions were not associated with apo E, even age varying from 38 to 79 years-old. However, the sample size and racial characteristics together with occlusive and aneurysmal patterns of atherosclerosis may have altered this result.

Regardless of the type of lesion, we conclude that this study on PAD was indifferent to apo E-Hha polymorphism. However, the apo E polymorphism may be associated to lesions of some types of peripheral arteries, being affected mainly by age, gender, and site of lesion, and this urges a study on sub-groups of patients with wider clinical cases.

REFERENCES

1. Shore VG, Shore B. Heterogeneity of human plasma very low density lipoproteins. Separation of species differing in protein components. Biochemistry 1973;12:502-50.

2. Zannis VI. Molecular biology of human apolipoprotein B and E and associated diseases of lipoprotein metabolism. Adv Lip Res 1989;23:2-64.

3. Strittmatter WJ. Apolipoprotein E and Alzheimer disease. Am Acad Neur 1994;4:444-5.

4. Mazzone T. Apolipoprotein E secretion by macrophages: its potential physiological functions. Curr Opin Lipidol 1996;7:303-7.

5. Utermann G, Hees M, Steinmetz A. Polymorphism of apolipoprotein E and occurrence of dysbetalipoproteinaemia in man. Nature 1977;269:604-7.

6. Zannis VI, Just PW, Breslow JL. Human apolipoprotein E isoprotein subclasses are genetically determined. Am J Hum Genet 1981;33:11-24.

7. Utermann G, Steinmetz A, Weber W. Genetic control of human apolipoprotein E polymorphism: comparison of one and two-dimensional techniques of isoprotein analysis. Hum Genet 1982;60:344-51.

8. Utermann G, Jaeschke M, Menzel J. Familial hyperlipoproteinemia type III. Deficiency of a specific apolipoprotein (apo EIII) in the very low density lipoproteins. FEBS Lett 1975;56:352-5.

9. Dominiczak MH. Apoliporoteins and lipoproteins in human plasma. In: Rifai N, Warnick GH, Dominiczak MH, editors. Handbook of Lipoprotein Testing. Washington (DC): AACC Press; 1997. p. 598.

10. Resnick HE, Rodriguez B, Havilik R, Ferruci L, Foley D, Curb JD, Harris TB. ApoE genotype, diabetes, and peripheral arterial disease in older men: the Honolulu Asia - aging study. Genet Epidemiol 2000;19:52-63.

11. Cattin L, Fisicaro M, Tonizzo M, et al. Polymorphism of the apolipoprotein E gene and early carotid atherosclerosis defined by ultrasonography in a asymptomatic adults. Arterioscler Thromb Basc Biol 1997;17:91-4.

12. Ilveskoski E, Perola M, Lehtimäki T, et al. Age-dependent association of apolipoprotein E genotype with coronary and aortic atherosclerosis in middle-age men. Circulation 1999;10:608-13.

13. Gustincich S, Manfioleti G, Del San G, Schiider C. A fast method for high-quality genomic DNA extraction from whole human blood. Bio Thechniques 1991;11:298-301.

14. Hixson JE, Vernier DT. Restriction isotyping of human apolipoprotein E by gene amplification and cleavage with Hha I. J Lipid Res 1990;31:545-8.

15. Scuteri A, Bos AJG, Zonderman AB, Brant LJ, Lakatta EG, Fleg JL. Is the apo 4 allele an independent predictor of coronary events? Am J Med 2001;10:28-32.

16. Davignon J, Gregg RE, Sing CF. Apoliprotein E polymorphism and atherosclerosis. Arteriosclerosis 1988;8:1-21.

17. Salazar LA, Hirata MH, Giannini SD, et al. Seven DNA polymorphism at the candidate genes of atherosclerosis in Brazilian women with angiographically documented coronary artery disease. Clin Chim Acta 2000;300:139-49.

18. Humpries SE, Talmud PJ, Hawe E, Bolla M, Day INM, Miller GJ. Apolipoprotein 4 and coronary heart disease in middle aged men who smoke: a prospective study. Lancet 2001;358:115-19.

19. Petrovic D, Zorc M, Peterlin B. Effect of apolipoprotein E polymorphism and apolipoprotein A-1 gene promoter polymorphism on lipid parameters and premature coronary artery disease. Folia Biol 2000;46:181-5.

20. Seo HS, Lombardi DM, Polinsky P, et al. Peripheral vascular stenosis in apolipoprotein E-deficient mice. Potential roles of lipid deposition, medial atrophy, and adventitial inflammation. Arterioscler Thromb Vasc Biol 1997;17:3593-601.

21. Vauhkonen I, Niskanen L, Ryynanen M, et al. Divergent association of apolipoprotein E polymorphism with vascular disease in patients with NIDDM and control subjects. Diabet Med 1997;14:748-56.

22. Gerdes LU, Lindholt JS, Vammen S, Henneberg EW, Fasting H. Apolipoprotein E genotype is associated with differential expansion rates of small abdominal aortic aneurysm. Br J Surg 2000;87:760-5.