Jin ZQ et al / Acta Pharmacol Sin 2004 Mar; 25 (3): 352-356

Association of apolipoprotein E 4 polymorphism with cerebral infarction in Chinese Han population¹

Zhu-qing JIN², Yong-sheng FAN³, Jing DING⁴, Mei CHEN⁵, Wei FAN⁴, Guang-ji ZHANG², Bin-hui ZHANG⁵, Suo-jing YU², Yong-sheng ZHANG², Wei-feng JI⁵, Jian-gang ZHANG⁶

²Genomic Research Centre, Zhejiang College of Traditional Chinese Medicine, Hangzhou 310053;
³School of Basic Medical Sciences, Zhejiang College of Traditional Chinese Medicine, Hangzhou 310053;
⁴Department of Neurology, Zhongshan Hospital, Fudan University, Shanghai 200032; ⁵Department of Neurology, Zhejiang Hospital of Traditional Chinese Medicine, Zhejiang College of Traditional Chinese Medicine, Hangzhou 310006, China

¹ Project supported by grant from Education Commission of Zhejiang province of China.

⁶ Correspondence to Dr Jian-gang ZHANG. E-mail zhangjg1126@yahoo.com

Received 2003-01-27 Accepted 2003-05-30

KEY WORDS apolipoproteins E; human; alleles; polymorphism; brain infarction

ABSTRACT

AIM: To study the association between APOE polymorphisms and cerebral infarction through a case-control study among the Chinese Han population. Methods: First-ever cerebral infarction patients (n=226) whose ages ranged from 40 to 60 years old were recruited from Department of Neurology, Zhongshan Hospital, Shanghai, and Zhejiang Chinese Traditional Medicine Hospital, Zhejiang, China. Unrelated healthy controls (n=201) were selected from the general population in the same area with similar age and sex distribution. APOE was amplified by one-stage PCR using the forward primer: 5'-GGC ACG GCT GTC CAA GGA GCT-3' and reverse primer: 5'-GAT GGC GCT GAG GCC GCG CT-3'. The PCR product was digested directly with 5 U of CfoI and separated by a 20 % polyacrylamide (acrylamide: bis-acrylamide=29:1) nondenaturing gel. Results: Both cerebral infarction patient and control groups were in Hardy-Weinberg equilibrium. The allele frequency of APOE*2, APOE*3, and APOE*4 was 4.6 %, 81.9 %, and 13.5 % respectively in the patients with cerebral infarction; 5.7 %, 87.3 %, and 7.0 % respectively in the healthy control group. Compared with APOE3/3 subjects, APOE4/4 carriers had a 2.1-fold risk of cerebral infarction (odds ratio 2.1, 95 % confidence limits 1.3 to 3.4). The allele frequency of APOE*4 in the cerebral infarction patient group was significantly higher than that in the control group (13.5 % vs 7.0 %; P=0.002). Conclusion: APOE 4 is a risk factor for cerebral infarction among the Chinese Han population.

INTRODUCTION

Stroke is one of the three leading causes of morbidity and mortality in both developing and developed countries^[1]. Genetic and environmental risk factors
relating to stroke have been studied in the past. The principal risk factors for stroke (including age and hypertension) are well defined, and genetic factors may account for some of this risk. For example, twin studies^[2,3], animal studies^[4], epidemiologic data^[5], and monogenic stroke disorders^[6] have provided the most clear evidence for genetic influences in stroke. It is believed that genes may play a role in the pathogenesis of stroke.

Apolipoproteins E (ApoE) is one of the major protein constituents in very low-density lipoprotein and plays a central role in lipid metabolism. It is the product of a single gene on chromosome 19 with the molecular weight of 34000 kDa glycoprotein which is composed of 299 amino acid residues^[7].

Although the association between APOE polymor-phism, in particular the relative proportion of APOE*4, and stroke-related pathologies have been investigated in Western populations, few publication can be found in international journals on the study of relationship between APOE polymorphism and cerebral infarction among the Chinese Han population, the largest population in the world, which easily offers a large sample size. The current study focused on the detailed studies of APOE polymorphism and cerebral infarction in the Chinese Han population.

In order to search for genetic influences on cerebral infarction, a kind of subtypes of stroke in Chinese Han, we studied a candidate gene, apolipoprotein E (APOE) which was controversial in the Western popu-lation.

MATERIALS AND METHODS

Subjects First-ever cerebral infarction patients (97 female and 129 male, total 226) whose ages ranged from 40 to 60 years old were recruited from Department of Neurology, Zhongshan Hospital, Fudan University, Shanghai, China and Zhejiang Traditional Hospital, Zhejiang College of Traditional Chinese Medicine, Hangzhou, Zhejiang, China, 147 km away from Shanghai. All the patients were systematically evaluated within 24 h of the symptom onset. The neurologist completed a detailed clinical questionnaire including information of vascular risk factors and physical examination of each patient. Electrocardiography, cardiac and carotid ultrasonography, and MR angiography are performed routinely for the cerebral infarction patients. The final diagnosis of cerebral infarction patients was confirmed by serial CT or MRI findings^[8].

Unrelated healthy controls (92 female and 109 male, total 201) were selected from the general population in the same area. They must meet the following criteria: good physical and mental health confirmed by clinical, radiological, and biological examinations.

A brief cognitive test^[9] was conducted to exclude any subject having potential cognitive impairment in both cerebral infarction patients and controls.

Peripheral blood samples (2 mL each) were collected in acid citrate dextrose (ACD)^[10] tubes after 12-h overnight fasting from all the control subjects. All blood samples were obtained within 24 h of stroke onset from cerebral infarction patients.

The study was started from September 1998 to August 2002 and was approved by the Hospital Ethics Committee and informed consent was obtained from all subjects.

A blinded APOE genotyping study was conducted including both cerebral infarction patients and control subjects.

Amplification of APOE gene from genomic DNA for restriction isotyping

Amplification of APOE gene from genomic DNA The protocol for isolation of genomic DNA from peripheral blood samples in this study was the same as that described by Russell^[11]. The amplification of APOE gene region encoding common APOE isoforms from genomic DNA was conducted in a DNA Thermal Cycler (Perkin Elmer Cetus 9600) using oligonucleotide primers (forward primer: 5'-GGC ACG GCT GTC CAA GGA GCT-3' and reverse primer: 5'-GAT GGC GCT GAG GCC GCG CT-3'). Each amplification reaction contained 100 ng of genomic DNA, 8 pmol of each primer, 5 mL of 10×PCR reaction buffer [0.67 mol/L Tris-HCl (pH 8.8), 0.166 mol/L (NH₄)₂SO₄, 0.067 mol/L MgCl₂, 0.72 % (v/v) b-mercaptoethanol (Sigma)], 10 % (v/v) dimethylsulfoxide (Sigma), 200 mmol/L of dATP, dTTP, dCTP, and dGTP (Merck), 2 U of Taq polymerase in a final volume of 50 mL. Each reaction mixture was denatured at 94 ºC for 2 min and subjected to five cycles of amplification by primer annealing (67 ºC for 10 s), extension (72 ºC for 80 s) and denaturation (94 ºC for 10 s). Immediately after completing this mini-cycle, the reaction mixture was continuously subjected to 30 cycles of amplification by primer annealing (60 ºC for 5 s), extension (72 ºC for 80 s), and denaturation (94 ºC for 10 s). The length of PCR product was 262 bp.

Analysis of amplified APOE product with RFLP After PCR amplification, 12 mL of each unpurified PCR product was directly digested with 5 U of CfoI at 37 ºC for overnight. The digested product was loaded onto a 20 % 1-mm thick and 7-cm long nondenaturing polyacrylamide gel (acrylamide:bis-acrylamide=29:1) for 2.5 h under constant voltage (150 V). After electropho-resis, the gel was treated with ethidium bromide (0.5 mg/L) for 6 min and DNA fragments were visualized by UV illumination. The sizes of CfoI fragments were determined by the marker^[12,13] (MspI-digested pUC18 DNA).

Although the total length of PCR product was different from that obtained by Hixon and Vernier due to the difference of reverse primer, the length for APOE typing was the same as Hixon since the surplus of the base pair was cut off at another CfoI cleavage site contained in the reverse primer.

Statistical analysis Allele frequencies were calculated by allele counting. Hardy-Weinberg equilibrium^[14] was tested by a c² goodness-of-fit test. The other data were compared by a  c² test. The computer software of Epi Info 6.04 edition was also used in the analysis. The Mantel-Haenszel^[15] method was used to calculate the adjusted odds ratio (OR) with 95 % confidence limits (CI).

RESULTS

The allele frequency of APOE*2, APOE*3, and APOE*4 was 4.6 %, 81.9 %, and 13.5 % respectively in the cerebral infarction patient group; 5.7 %, 87.3 %, and 7.0 % respectively in the healthy control group. Compared with APOE3/3 subjects, 4 carriers had a 2.1-fold risk of cerebral infarction (OR 2.1, 95 % CI 1.3 to 3.4). The allele frequency of APOE*4 in the cerebral infarction patient group was significantly higher than that in the control group (13.5 % vs 7.0 %; P=0.002; Tab 1).

Tab 1. Distribution (n, subjects number)of APOE allele genotype in both cerebral infarction patient and control groups. ^bP<0.05 vs control.

APOE	Cerebral Infarction			Control
Geno-	Observed Expected		c2	Observed Expected		c2
type
2/2	2	0.4878	4.688	2	0.658	2.737
2/3	14	17.19	0.592	17	20.08	0.4724
2/4	3	2.834	0.0097	2	1.602	0.0989
3/3	152	151.4	0.0036	156	153.2	0.0512
3/4	52	49.93	0.0095	22	24.45	0.2455
4/4	3	4.116	0.3026	2	0.9751	1.077
Total	226	226	5.605	201	201	4.682

The allele and genotype frequencies were in Hardy-Weinberg equilibrium by comparison with that of the corresponding theoretical distribution. Both cerebral infarction patient and control groups were in Hardy-Weinberg equilibrium (P>0.05 respectively; df=5; Tab 2).

Tab 2. Check for Hardy-Weinberg equilibrium in both cerebral infarction patient and control groups. c²=5.605 and 4.682, respectively, P>0.05. df=5. Age, Mean±SD.

	Cerebral infarction	Control
	(n=226)	(n=201)
Sex (F/M)	97/129	92/109
Age/a	48.5¡À3.4	47.1¡À2.4
E2/2	2	2
E2/3	14	17
E2/4	3	2
E3/3	152	156
E3/4	52	22
E4/4	3	2
E*2	21 (4.6 %)	23 (5.7 %)
E*3	370 (81.9 %)	351 (87.3 %)
E*4	61 (13.5 %)b	28 (7.0 %)

DISCUSSION

The APOE allele frequencies have been found to be associated with some kind of diseases. Three common alleles of APOE are APOE*2, APOE*3, and APOE*4^[16,17], which can be distinguished by the variation of positions at 112 and 158 in the receptor-binding region of ApoE^[18]. If positioned with 112 cys and 158 arg, it forms isoform APOE*3 which is the commonest isoform; If the positioned with 112 arg and 158 arg, it forms isoform APOE*4 which is associated with Alzheimer's disease^[19] , vascular dementia^[20], cognition in the very old^[21], artery disease^[22], gallstones^[23], diabetes, and atherosclerosis^[24], colon cancer^[25], etc; If positioned with 112 cys and 158 cys, it forms isoform APOE*2 which is a risk factor for type III hyperlipidemia^[26].

Our data shows significantly higher frequency of APOE*4 allele in the Chinese Han patient with cerebral infarction when compared with the control group; and this is similar to the results in the studies of ischemic stroke by Pedro-Botet et al^[27] in 100 males in the Spanish and Margaglione et al^[28] in the Italian population, Peng et al^[29] in the Chinese population, Kessler et al^[30] in the German population. However, our results do not show the significantly higher frequency of APOE*2 allele as evidenced in a Japanese rural population^[31], Couderc et al^[32], Ferrucci et al^[33] in a cohort study. Basun et al^[34], Catto et al^[35], Kuusisto et al^[36], MacLeod et al^[37], Morrison et al^[38], and Nakata et al^[39] found no association between APOE and cerebral infarction. Surprisingly a study by McCarron and colleagues found a favourable effect of APOE4 on stroke outcome^[40]. All these controversial data, firstly, may be mainly due to the ethnic difference; secondly, might be the different criteria for selecting the unrelated healthy subjects as controls since many diseases in the control group could not be easily ruled out especially for those early cognitive impaired healthy subjects. For this reason, all the healthy subjects were given a brief cognitive test in our study.

Interestingly, the case-control study showed inconsistency data, but the animal study presented promising results. Laskowitz et al^[41] utilized apolipoprotein E-deficient mice as a model, and found that apolipopro-tein E had increased susceptibility to focal cerebral ischemia. Furthermore, Sheng H et al^[42] used transgenic mice model, and detected that apolipoprotein E3 was favorable for focal ischemia when it was compared to apolipoprotein E4 in transgenic mice. These data suggested that the epsilon 4 allele could increase infarct size and functional impairment; and they also supported that APOE might be a key molecule for the clearance of infarction tissue after ischemia in the brain.

The study of gene-environment and gene-gene interactions, as well as genotype to phenotype links, is critical in order to gain insight into complex traits. The recent advances in the human genome project^[43,44] and the identification of multiple single-nucleotide polymorphisms within the human genome^[45], combined with bioinformatics, will facilitate the study of multifactorial, complex disorders such as stroke. Finally, the identification of causative genes will facilitate early diagnosis, prevention, and a more effective treatment of ischemic stroke.

REFERENCES

• 1 Stegmayr B, Asplund K, Kuulasmaa K, Rajakangas AM, Thorvaldsen P, Tuomilehto J. Stroke incidence and mortality correlated to stroke risk factors in the WHO MONICA Project: an ecological study of 18 populations. Stroke 1997; 28: 1367_74.
• 2 Brass LM, Isaacsohn JL, Merikangas KR, Robinette CD. A study of twins and stroke. Stroke 1992; 23: 221-3.
• 3 Hrubec Z, Robinette CD. The study of human twins in medical research. N Engl J Med 1984; 310: 435-41.
• 4 Coyle P, Odenheimer DJ, Sing CF. Cerebral infarction after middle cerebral artery occlusion in progenies of spontaneously stroke-prone and normal rats. Stroke 1984; 15: 711-6.
• 5 Kiely DK, Wolf PA, Cupples LA, Beiser AS, Myers RH. Familial aggregation of stroke. The Framingham Study. Stroke 1993; 24: 1366-71.
• 6 Tournier-Lasserve E, Joutel A, Melki J, Weissenbach J, Lathrop GM, Chabriat H, et al. Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy maps to chromosome 19q12. Nat Gen 1993; 3: 256-9.
• 7 Mahley RW. Apolipoprotein E: cholesterol transport protein with expanding role in cell biology. Science 1988; 240: 622-30.
• 8 Special report from the National Institute of Neurological Disorders and Stroke: classification of cerebrovascular diseases III. Stroke 1990; 21: 637-76.
• 9 Zhang MY, Katzman R, Salmon D, Jin H, Cai GJ, Wang ZY, et al. The prevalence of dementia and Alzheimer's disease in Shanghai, China: impact of age, gender and education. Ann Neurol 1990; 27: 428-37
• 10 Sambrook J, Fritsch EF, Maniatis T. In: Molecular cloning (2^nd ed. Chinese ed). New York: Cold Spring Harbor Laboratory Press; 1989. p 465-6.
• 11 Russell H, 1989. Simple and rapid preparation of samples for PCR. In: Erlich HA, editor. New York: PCR Technology Stockton Press; 1989. p 31-8.
• 12 Hixon JE, Vernier DT. Restriction isotype of human apolipoprotein E by gene amplification and cleavage with HhaI. J Lipid Res 1990; 31: 545-8.
• 13 Wenham PR, Price WH, Blundell G. Apolipoprotein E genotyping by one-stage PCR. Lancet 1991; 337: 1158-9.
• 14 Hartl DL. Principles of population genetics. Sinauer: Sunderland, Mass; 1980.
• 15 Mantel N, Haenszel W. Statistical aspects of the analysis of data from retrospective studies of disease. J Natl Cancer Inst 1959; 22: 719-48.
• 16 Utermann G, Langenback U, Beisiegel U, Weber W. Genetics of the apolipoprotein E system in man. Am J Hum Genet 1980; 32: 339-47.
• 17 Zannis VI, Breslow JL. Human VLDL apoE isoprotein polymorphism is explained by genetic variation and post-translational modification. Biochemistry 1981; 20: 1033-41.
• 18 Weisgraber KH, Rall SC Jr, Mehley RW. Human E apoprotein heterogeneity: cysteine-arginine interchanges in the amino acid sequence of the apoE isoforms. J Biol Chem 1981; 256: 9077-83.
• 19 Strittmatter WJ, Roses AD. Apolipoprotein E and Alzheimer's disease. Annu Rev Neurosci 1996; 19: 53-77.
• 20 Shigeji N, Kaori M, Nobuhiro Y. Apolipoprotein E genotype and Alzheimer's disease. Lancet 1993; 342: 737.
• 21 Altstiel LD, Greenberg DA, Marin D, Lantz M, Mohs R. Apolipoprotein E genotype and cognition in the very old. Lancet 1997; 349: 1451.
• 22 Moore JH, Reilly SL, Ferrell RE, Sing CF. ApoE polymorphism may be a gender-specific predirector of coronary artery disease age of onset. Clin Genet 1997; 51: 22-5.
• 23 Bertomeu A, Ros E, Zambon D, Vela M, Perez-Ayuso RM, Targarona E, et al. Apolipoprotein E polymorphism and gallstones. Gastroenterology 1996; 111: 1603-10
• 24 Semenkovich CF, Heinecke JW. The mystery of diabetes and atherosclerosis: time for a new plot. Diabetes 1997; 46: 327-34.
• 25 Kervinen K, Sodervik H, Makela J, Lehtola J, Niemi M, Kairaluoma MI, et al. ApoE polymorphism: another player in the genetics of colon cancer susceptibility? Gastroenterology 1996; 110: 1785-90.
• 26 Breslow JL, Zannis VI, SanGiacomo TR, Third JL, Tracy T, Glueck CJ. Studies of familial type III hyperlipoproteinemia using as a genetic marker the apoE phenotype E2/2. J Lipid Res 1982; 23: 1224-35.
• 27 Pedro-Botet J, Senti M, Nogues X, Rubies-Prat J, Roquer J, D'Olhaberriague L, et al. Lipoprotein and apolipoprotein profile in men with ischemic stroke: role of lipoprotein(a), triglyceride-rich lipoproteins, and apolipoprotein E polymorphism. Stroke 1992; 23: 1556-62.
• 28 Margaglione M, Seripa D, Gravina C, Grandone E, Vecchione G, Cappucci G, et al. Prevalence of apolipoprotein E alleles in healthy subjects and survivors of ischemic stroke: an Italian case-control study. Stroke 1998; 29: 399-403.
• 29 Peng DQ, Zhao SP, Wang JL. Lipoprotein (a), and apolipoprotein E 4 as independent risk factors for ischemic stroke. J Cardiovasc Risk 1999; 6: 1-6.
• 30 Kessler C, Spitzer C, Stauske D, Mende S, Stadlmuller J, Walther R, et al. The apolipoprotein E and b-fibrinogen G/A-455 gene polymorphisms are associated with ischemic stroke involving large-vessel disease. Arterioscler Thromb Vasc Biol 1997;17: 2880-4.
• 31 KokuboY, Chowdhury AH, Date C, Yokoyam T, Sobue H, Tanaka H. Age-dependent association of apolipoprotein E genotypes with stroke subtypes in a Japanese rural popula-tion. Stroke 2000; 31: 1299-306.
• 32 Couderc R, Mahieux F, Bailleul S, Fenelon G, Mary R, Fermanian J. Prevalence of apolipoprotein E phenotypes in ischemic cerebrovascular disease: a case-control study. Stroke 1993; 24: 661-4.
• 33 Ferrucci L, Guralnik JM, Pahor M, Harris T, Corti MC, Hyman BT, et al. Apolipoprotein E 2 allele and risk of stroke in the older population. Stroke 1997; 28: 2410-6.
• 34 Basun H, Corder EH, Guo Z, Lannfelt L, Corder LS, Manton KG, et al. Apolipoprotein E polymorphism and stroke in a population sample aged 75 years or more. Stroke 1996; 27: 1310-5.
• 35 Catto AJ, McCormack LJ, Mansfield MW, Carter AM, Bamford JM, Robinson P, et al. Apolipoprotein E polymorphism in cerebrovascular disease. Acta Neurol Scand 2000;101: 399-404
• 36 Kuusisto J, Mykkanen L, Kervinen K, Kesaniemi YA, Laakso M. Apolipoprotein E4 phenotype is not an important risk factor for coronary heart disease or stroke in elderly subjects. Arterioscler Thromb Vasc Biol 1995; 15: 1280-6.
• 37 MacLeod MJ, De Lange RP, Breen G, Meiklejohn D, Lemmon H, Clair DS. Lack of association between apolipoprotein E genoype and ischaemic stroke in a Scottish population. Eur J Clin Invest 2001; 31: 570-3.
• 38 Morrison AC, Ballantyne CM, Bray M, Chambless LE, Sharrett AR, Boerwinkle E. LPL polymorphism predicts stroke risk in men. Genet Epidemiol 2002; 22: 233-42.
• 39 Nakata Y, Katsuya T, Rakugi H, Takami S, Sato N, Kamide K, et al. Polymorphism of angiotensin converting enzyme, angiotensinogen, and apolipoprotein E genes in a Japanese population with cerebrovascular disease. Am J Hypertens 1997; 10: 1391-5.
• 40 McCarron MO, Muir KW, Weir CJ, Dyker AG, Bone I, Nicoll JA, et al. The apolipoprotein E e4 allele and outcome in cerebrovascular disease. Stroke 1998; 29: 1882-7.
• 41 Laskowitz DT, Sheng H, Bart RD, Joyner KA, Roses AD, Warner DS. Apolipoprotein E-deficient mice have increased susceptibility to focal cerebral ischemia. J Cereb Blood Flow Metab 1997; 17: 753-8.
• 42 Sheng H, Laskowitz DT, Bennett E, Schmechel DE, Bart RD, Saunders AM, et al. Apolipoprotein E isoform-specific differences in outcome from focal ischemia in transgenic mice. J Cereb Blood Flow Metab 1998; 18: 361-6.
• 43 Lander ES, Linton LM, Birren B, Nusbaum C, Zody MC, Baldwin J, et al. Initial sequencing and analysis of the human genome. Nature 2001; 409: 860-921.
• 44 Venter JC, Adams MD, Myers EW, Li PW, Mural RJ, Sutton GG, et al. The sequence of the human genome. Science 2001; 291: 1304-51.
• 45 Hirakawa M, Tanaka T, Hashimoto Y, Kuroda M, Takagi T, Nakamura Y. JSNP: a database of common gene variations in the Japanese population. Nucleic Acids Res 2002; 30.