Genetic risk factors and ischaemic cerebrovascular disease: role of common variation of the genes encoding apolipoproteins and angiotensin-converting enzyme

224

+ O R I G I N A L A R T I C L E +

Genetic risk factors and ischaemic cerebrovascular disease: role of common

variation 4 of the genes encoding apolipoproteins and angio t ensin-converting enzyme

Katriina Aalt~-Setala'~~, Heikki Palomaki2, Helena Miettinen', Alpo Vuorio', Timo Kuusi', Raili Raininko 3, Oili Saloy2en3,

Markku Kaste2 and Kimmo Kontula'

DNA polymorphisms in genes encoding apolipoproteins (apo) A-I, C-111, B and E and angiotensin-converting enzyme (ACE) have been proposed to be associated with the risk of coronary artery disease (CAD). We studied whether the same genetic markers would also be associated with the occurrence and extent of atherosclerosis in cervical arteries. DNA samples from 234 survivors of stroke or a transient ischaemic attack aged 60 years or less were examined. The presence of atherosclerosis was assessed using aortic arch angiograms. The SstI polymorphism of apoA-VC-I11 gene locus, the XbaI polymorphism of apoB gene, common apoE phenotypes and the insertion/deletion polymorphism of the ACE gene were analysed. The allele frequencies of the apoA-I/C-111, apoB, apoE or ACE gene did not differ between the groups with ( n = 148) or without ( n = 85) cervical atherosclerosis. However, when patients with at least one apoE4 allele and one X2 allele of apoB were combined and compared with those without either of them (E2E3 or E3E3 and X l X l ) , a significant association with the presence of cervical atherosclerosis was found (P = 0.03). The patients having the E2E3 phenotype had a significantly elevated serum triglyceride level compared with those with the E3E3 phenotype (P = 0.03). Serum high-density lipoprotein (HDL) cholesterol was lower in the patients with the E2E3 phenotype than in those with the E3E3 and E3E4 ( P = 0.01 and P = 0.06, respectively). The apoB or ACE genotypes were not significantly associated with serum lipid or lipoprotein levels. There was no association between the ACE gene polymorphism and the occurrence of hypertension. In conclusion, the interaction of common apoB and apoE alleles may increase the risk of atherosclerosis in cervical arteries.

Key words: apolipoproteins; angiotensin-converting enzyme; ischaemic stroke; transient ischaemic attact.

Ann Med 1998; 30: 224-233.

Introduction From the Departments of 'Medicine, *Neurology and

'Radiology, University of Helsinki, Helsinki, Finland and the 4Department of Medicine, University of Tampere, Tampere, Finland.

Correspondence: Katriina Aalto-SetCli, MD, Department of Medicine, Tampere University Hospital, PO Box 2000, FIN-33521 Tampere, Finland. E-mail: [email protected], Fax +358 3 2474362.

Received on 21 August, 1997; revision accepted on 9 December, 1997.

Stroke is among the most common causes of mortality and morbidity in industrial countries (1, 2). Stroke primarily affects elderly people, but about 20% of strokes occur before the age of 65 (2, 3). In a World Health Organization (WHO) collaborative study involving 17 centres, the highest incidences of stroke were recorded in Japan and Finland (4). This finding

0 The Finnish Medical Society Duodecim, Ann Med 1998; 30: 224-233

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GENETIC RISK FACTORS AND ISCHAEMIC B R A I N DISEASE 225

was confirmed in subsequent studies ( 5 ) , even though the incidence of coronary heart disease (CHD) differs significantly in these two countries (6). Established risk factors for stroke include advanced age, male sex, hypertension, cardiac diseases, diabetes mellitus and smoking (7, 8). Studies on the association of dyslipidaemias with stroke have revealed contradictory results. Epidemiological studies have failed to demonstrate a significant association, but studies with defined types of cerebrovascular atherosclerosis have disclosed correlations between serum lipids or lipoproteins and atherosclerosis (for review, see (9)). Thus, elevated serum total or low-density lipoprotein (LDL) cholesterol, elevated serum triglycerides, decreased high-density lipoprotein (HDL) cholesterol, increased serum lipoprotein (a) (Lp(a)) or their combinations have all been related to cerebrovascular disease. Familial aggregation of stroke (10) strongly favours the contribution of genetic factors. The exact nature of the underlying genes remains, however, totally unknown, as is their possible relationship with the risk factors cited above.

A number of genetic markers such as the SstI polymorphism of the apolipoprotein (apo) A-I/C-111 gene locus, the XbaI polymorphism of the apoB gene, and the common three-allelic polymorphism of the apoE gene have been found to be associated with dyslipidaemias and an increased risk of CHD (for review, see (11, 12)). ApoA-I is the major structural protein of HDL, and its plasma level correlates inversely with the incidence of CHD (13, 14). ApoC- I11 is present in chylornicrons, very-low-density lipoproteins (VLDL) and HDL, and it is known to affect triglyceride metabolism (15, 16). An SstI restriction fragment length polymorphism located in the 3’-noncoding region of the apoA-I/C-I11 gene complex has been found to associate with hypertriglyceridaemia ( 17-22) and an increased risk of coronary artery disease (23,24) in many populations. ApoB is a large protein needed for the assembly of triglyceride-rich lipoproteins, and it is also responsible for the interaction of LDL with its cell membrane receptor. The XbaI DNA polymorphism of the apoB gene has been found to be associated with serum cholesterol levels in random populations (25- 27) and in patients with familial hypercholes- terolaemia (FH) (28) as well as with the susceptibility to coronary artery disease (CAD) (29). ApoE is an important regulator of the lipoprotein metabolism, having roles in the removal of triglyceride-rich lipoproteins from the circulation, in the conversion of VLDL to LDL and in the ‘reverse cholesterol transport’ trafficking cholesterol from peripheral tissues to liver. ApoE exists in three different isoforms (E2, E3 and E4) encoded by three different alleles ( ~ 2 , ~3 and ~ 4 ) . Several studies have indicated that carriers of the ~4 allele have elevated serum LDL-cholesterol

levels (30-32) and an increased risk of CHD (33, 34). Another gene variation suggested to play a role as a regulator of the risk of myocardial infarction is the insertion/deletion polymorphism of the anziotensin- converting enzyme (ACE) gene (35) although the mechanism involved remains poorly understood.

The present study was carried out in order to find out whether any of the common gene polymorphisms proposed to be related to the risk of CHD would also be associated with the occurrence and extent of atherosclerotic cerebrovascular disease. To this end, we analysed the apolipoprotein and ACE gene polymorphisms in a cohort of 234 survivors of ischaemic stroke or a transient ischaemic attack (TIAj. Allele frequencies were related to the findings on aortic arch angiograms.

Methods

Patients

Blood samples were taken from 234 patients with an ischaemic stroke or TIA. These patients were recruited by an invitation to an initial cohort of 423 consecutive and relatively young (age 5 60 years) patients admitted at the Department of Neurology, University of Helsinki, during a 3-year period. Patients using lipid- lowering drugs or who, according to angiographic studies, had arterial diseases unrelated to atherosclerosis were excluded. The medical, smoking and family history of 294 patients have been reported earlier (36), and 234 of them participated in the DNA analysis. The clinical data of the patients in the present study did not differ from those published earlier. Hypertension was considered to be present if a previous diagnosis of essential hypertension had been set and the patient used antihypertensive drugs, or if the patient had sustained systolic blood pressure values of at least 150 mmHg or diastolic blood pressure values of at least 100 rnmHg during the study.

Angiography

The presence of any visible atherosclerotic lesions in 11 extracranial arteries (brachiocephalic, subclavian, vertebral, common carotid and internal and external carotid arteries) in the aortic arch angiograms was assessed by two neuroradiologists without knowledge of the patient history. To reach a uniform grading of angiographic atherosclerosis, the first 20 angiograms were simultaneously evaluated by both observers. They worked closely together also at the later stage, and all initial gradings considered doubtful by one observer were reviewed also by the other, and a consensus grading was reached. Earlier, the interobserver correlation in evaluating stenotic lesions


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226 AALTO-SETALA PALOMAKI MIETTINEN ET AL

in cervicocranial arteries has been good (37) as has also been the reliability of the same measurements by one observer at different time points (37). The measurements were commenced at the aortic arch, and all extracranial parts of the arteries were evaluated. Both the anteroposterior and the left oblique views of the angiograms were evaluated. In two patients, anteroposterior projections of the angiograms were not available, leaving 232 for futher analysis. For the presence of atherosclerosis, all visible atherosclerotic lesions in both projections of the angiograms were taken into account. The thickness and the length of the atherosclerotic lesions were measured, as well as the percentage of stenosis caused by the plaques. The sum of the percentage of stenoses in the arteries was computed, which implies that the sum of the percentage of stenoses exceeded 100 in patients with severe stenosis. If any atherosclerosis was observed in the angiograms, the patient belonged to the group of ‘atherosclerosis present’. Without any visually observed atherosclerosis the patient was assigned to the ‘atherosclerosis absent’ group.

Serum lipids and lipoproteins

The median time interval between stroke or TIA and the collection of blood samples was 11 months (range 2.5-35 months). Acute samples were avoided to exlude any influence of acute cerebrovascular events on serum lipid values (38). Blood samples were taken in the morning after the patients had fasted for 12 h. Lipoprotein fractions were separated by sequential ultracentrifugation (39). Serum total cholesterol concentration as well as cholesterol in the LDL and HDL fractions were determined by a commercial kit (Boehringer Mannheim, Mannheim, Germany). Triglyceride concentrations in the whole blood and in the VLDL fraction were also determined using a commercial kit (Boehringer Mannheim, Mannheim, Germany).

D N A analysis

DNA was isolated from 10 mL of EDTA-treated venous blood, and digested with the restriction enzymes SstI and XbaI for apoA-IIC-I11 and apoB analysis, respectively. The presence or absence of the polymorphic cutting sites was visualized using Southern blotting analysis. The X2 and S2 alleles denote alleles with the polymorphic cleavage site present, and the X1 and S1 alleles lack the polymorphic site. The insertion (i)/deletion (d) polymorphism of intron 16 of the ACE gene was determined by a polymerase chain reaction (PCR) assay according to Rigat and co-workers (40), with

confirmation of the dd genotype by the technique described by Shanmugam and co-workers (41).

Analysis of apoE phenotypes

ApoE phenotyping was carried out by analytical isoelectric focusing on polyacrylamide gel containing 8 mol/L urea and 2% Ampholine, pH 4-6 (LKB, Bromma, Sweden). VLDL and LDL fractions (d < 1.019) were separated by ultracentrifugation (39). The delipidation of the samples was performed as described earlier (42), and isoelectric focusing was carried out by standard techniques (30).

Allele frequencies in the population Data on the apoA-I/C-111 ( n = 61) and apoB ( n = 176) gene polymorphisms in a random healthy population (mean age 40 years, range 18-66 years) were derived from our previous studies (21, 30). Frequencies of the apoE phenotypes in 615 healthy Finnish blood donors (age 20-55 years) were obtained from the study of Ehnholm et a1 (30). All these population samples came from cohorts representing the same residence area (Helsinki and its neighbouring area) as the patients with cerebrovascular disease.

Working ability a t the follow-up The working ability of the patients was clarified by a simple questionnaire (1 = currently working; or 2 = currently on sick leave or retired) at the time of sample collection.

Statistical analysis Statistical analysis of the data was performed using the BMDP statistical software package (BMDP Statistical Software Inc, Los Angeles, CA, USA). When comparing genotype destributions and allele frequencies in different groups, X2-test or Fisher’s exact test was used. The differences in lipid values or in the degree of atherosclerosis between separate groups were compared using Mann-Whitney U-test. To clarify the independency of associations between certain genetic groups and atherosclerosis, a multiple logistic regression analysis was carried out. ANOVA was used in assessing the connection between the severity of atherosclerosis and the genetic groups.

Resu I t s

Of the 234 patients (135 males and 99 females), 140 (59.8%) had suffered an ischaemic stroke while 94 (40.2%) had symptoms compatible with TIA.


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GENETIC RISK FACTORS AND ISCHAEMIC BRAIN DISEASE 227

According to the evaluation of the 11 extracranial arteries, arteriosclerosis was present in 131 (56.5%) of the 232 anteroposterior projections, in 139 (59.4%) of the 234 oblique projections, and in 148 (63.2%) of the 234 patients when both projections were taken into account. The mean plaque thickness in the patients with atherosclerosis was 5.15 mm (range 0.25-28.8 mm), and their mean length was 47.7 mm (range 1.0-569.2 mm). The mean percentage of luminal stenosis was 87 percentage units (range 3-449 percentage units).

The SstI polymorphim in the 3'-noncoding region of the apoC-I11 gene was determined in 233 patients. The frequencies of the S2 allele (the polymorphic site present) and the S1 allele (the polymorphic site absent) were 11.6% and 88.4%, respectively, in the total study population. No statistically significant differences were observed between the allelic frequences of the apoA-I/ C-I11 gene locus in patients with or without atherosclerosis (Table 1). The XbaI polymorphism of the apoB gene, corresponding to a silent point

Table 1. Genotype distributions (number of subjects) and allele frequencies of the apoA-I/C-I11 and apoB gene loci, and apoE phenotype allele frequencies in patients with or without cerebrovascular atherosclerosis. The corresponding allele frequencies in healthy Finnish blood donors are also given.

Genetic Cerebrovascul atherosclerosis Blood group donors*

Patients Absent Present n n (YO) n (YO)

Ap~A- l /C- l l l S l S l s1 s 2 s2s2

Allele S1 (%) Allele S2 (%)

X lX l x1 x2 x2x2

Allele X1 (%) Allele X2 (%)

E2E2 E2E3 E2E4 E3E3 E3E4 '

E4E4 Total

Allele E2 (%) Allele E3 (%) Allele E4 (%)

Total

ApoB

Total

ApoE

185 42

6 233

0.884 0.116

94 106 33

233 0.631 0.369

1 13 2

135 67 13

231 0.037 0.758 0.206

67 (78.8) 15 (17.6) 3 (3.5)

85 (100) 0.88 0.12

37 (43.5) 33 (38.8) 15 (17.6) 85 (100) 0.62 0.38

0 (0) 3 (3.6) 1 (1.2)

57 (68.7) 14 (16.9) 8 (9.6)

83 (100) 0.02 0.79 0.19

118 (79.7)

3 (2.0) 27 (18.2)

148 (100) 0.89 0.92 0.1 1 0.08

57 (38.5) 73 (49.3) 18 (12.2)

148 (100) 0.63 0.58 0.37 0.42

1 (0.7) 10 (6.8) 1 (0.7)

78 (52.7) 53 (35.8) 5 (3.4)

148 (100) 0.04 0.06 0.75 0.70 0.22 0.24

*See Materials and Methods.

mutation at codon 2488, was also studied in 233 patients. The frequencies of the X2 allele (the polymorphic site present) and the X I allele (the polymorphic site absent) were 36.9% and 63.1%, respectively. The allelic frequencies were not significantly different in those with atherosclerosis compared with those without angiographically detectable cerebrovascular atherosclerosis (Table 1). The allele frequencies of the apoA-I/C-I11 and apoB loci were very similar among random blood donors and patients with cerebrovascular disease (Table 1).

The apoE phenotypes were determined from 231 patients. There was only one patient with the E2E2 phenotype and two patients with the E2E4 phenotype; these patients were excluded from further analysis on the association of apoE phenotypes with atherosclerosis and serum lipids. The apoE allelic frequencies were not significantly different in the patients with atherosclerosis compared with those without atherosclerosis (Table 1). However, the patients with the phenotype E3E4 had significantly more often atherosclerosis than the patients with other phenotypes ( P = 0.002), and the patients with the E3E3 phenotype had significantly less atherosclerosis than those with other phenotypes ( P = 0.02). The E3 allele occurred slightly more often in the patients with brain infarction than in random blood donors (76% vs 70%) (Table 1).

The serum lipid and lipoprotein levels in the patients with different apoA-I/C-I11 and apoB genotypes and apoE phenotypes are summarized in Table 2. The patients with the S2S2 genotype of the apoA-I/C-I11 gene complex tended to have a lower serum HDL-cholesterol concentration than those with the SlSl genotype ( P < 0.1). With apoE, those with the E2E3 phenotype had lower serum HDL-cholesterol levels than those with the E3E3 or E3E4/E4E4 phenotypes (P = 0.01 and P = 0.06, respectively), and serum triglyceride levels were significantly elevated in patients with the E2E3 phenotype compared with those with the E3E3 phenotype ( P = 0.03, Table 2). There was a trend towards higher serum total and LDL-cholesterol levels in patients with the E3E4/E4E4 phenotypes than in those with the E2E3 or E3E3 phenotypes, but these differences did not reach statistical significance (Table 2). In contrast, no significant differences were observed between any of the apoB XbaI genotypes and serum lipid or lipoprotein parameters.

The severity of atherosclerotic lesions in patients with different apoA-I/C-I11 and apoB genotypes and apoE phenotypes is presented in Table 3. Only patients with present atherosclerosis were included. Using ANOVA, no significant differences were observed in the apoA-I/C-I11 or apoB genotypes, or in the apoE phenotypes and in the plaque length or depth or percentage of stenosis (Table 3).


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228 AALTO-SETAI A PALOMAKl MlFTTlNFN FT A 1

Table 2. Serum lipids and lipoproteins (mmol/L, mean f SD) in patients with different apoA-I/C-III and apoB genotypes and apoE phenotypes.

Genetic Patients Cholesterol HDL LDL Trigly- group n cholesterol cholesterol cerides

ApoA-I/C-l 11 SlS1 185 6.42 2 1.37 1.51 k 0.41 4.49 * 1.22 1.64 k 0.94

1.66 k 0.71 s1s2 42 6.54 & 1.31 1.47 2 0.36 4.62 k 1.10 1.74 i 0.65 s2s2 6 6.09 + 1.45 1.28 k 0.36 4.40 k 1,41

ApoB x1 X I x1 x2 x2x2

94 6.33 k 1.33 1.48 k 0.39 4.45k1.18 1.60 2 0.77 106 6.54 2 1.43 1.52 2 0.42 4.60 k 1.29 1.67 I 1.01 33 6.39 k 1.21 1.48 2 0.37 4.44 * 0.99 1.71 k 0.89

ApoE 2.01 2 0.86 E2E3 13 6.18k1.97 1.26 k 0.23 4.19k0.36

E3E3 135 6.39 k 1.41 1.53 i 0.41' 4.47 k 1.26 1.57 0.80* E3E4/E4E4 80 6.54 k 1.24 1.47 0.40 4.64k1.12 1.70 2 1.04

* P < 0 05 when patients with E2E3 are compared with those wlth E3E3

Table 3. Severity of atherosclerotic lesions (in mm or in percentages) in patients with different apoA-T/C-lII and apoB genotypes and apoE phenotypes.

Genetic Plaque Patients Plaque Patients Stenosis, Q/~ Patients group tength (mm) n depth (mm) n (mean & SO) n

(mean & SD) (mean _t SD)

Ap~A- l /C- l l l s1s1 s1s2 5252

x1x1 x1 x2 x2x2

E2E3 E3E3 E3E4/E4E4

ApoB

ApoE

5 0 . 0 9 ~ 88.79 35.45+ 44.06 6 9 . 7 2 i 60.49

55.35+ 93.39 35.031 46.35 71.66 & 130.62

37.152 61.61 47.592 81.28 4 7 . 4 3 i 86.39

113 27

3

57 68 18

10 76 55

5.28 k 6.08 4.43k4.16 6.62 5 4.46

5.62 k 6.70 4.84 k 5.00 4.86 k 5.1 1

4.96 k 5.84 5.08 & 5.60 4.87 L 5.36

113 27 3

57 68 18

10 76 55

90.1 101.8 70 .22 64.0

140 k124.7

93.9 2 105.3 81 .02 91.7 92.1 + 87.7

110 k127 .6 8 5 . 2 ~ 94.1 79.1 k 89.2

113 27

3

57 71 18

10 78 56

In order to reveal a possible interaction of the different apolipoprotein genotypes and the risk of atherosclerosis, the patients with a t least one apoE4 allele and either at least one apoB X1 or X2 allele were compared with those without either of these alleles. No significant differences were observed in the risk of atherosclerosis when those with the apoE phenotype of E3E4 or E4E4 and the apoB genotype of XlX1 or X1X2 were compared with those with the apoE phenotype of E2E3 or E3E3 and the apoB genotype of X2X2 (data not shown). The data obtained from the patients with at least one apoE4 allele and at least one apoB X2 allele compared with those without either of these alleles are presented in Table 4. A nonsignificant trend towards elevated serum total and LDL-cholesterol levels was observed in the former genotype category (Table 4). Analysis of

the individual numerical dimensions of the atherosclerotic lesions favoured stronger angiographic evidence of atherosclerosis in carriers of both the E4 and X2 genes than in those lacking them (Table 4). When classified on an all-or-nothing basis, the patients with both the E4 and X2 alleles had atherosclerosis significanly more often than those without either of these alleles ( P = 0.03, Table 4). To estimate whether this gave any extra information over and above that associated with differences in lipids, a multiple logistic regression analysis was carried out with this genotype combination, cholesterol, HDI, cholesterol, LDL cholesterol and triglycerides as independent variables, and the presence of atherosclerosis as the dependent variable. Only the genotype and triglycerides remained in the logistic model as significant independent determinants of atherosclerosis. The odds ratio


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GENETIC RISK FACTORS AND

Table 4. Serum lipids and lipoproteins (mmol/L, mean k SD) and atherosclerotic lesions in patients with at least one E4 allele of the apoE gene and at least one X2 allele of the apoB gene, and in those without either of these alleles (n = number of patients).

Variable E3fE4 or E4fE4 E2fE3 or E3lE3 P X1X2 or X2X2 x1 x1

(n = 43) (n = 54)

Lipids Cholesterol HDL cholesterol LDL cholesterol Triglycerides

Lesions Length Depth Narrowing, %

Atherosclerosis Absent Present

6 . 5 2 ~ 1.29 1 . 4 7 ~ 0.37 4.61 L 1.11 1 . 7 7 ~ 1.23

28.95 50.42 3.69k 4.65

59.04 2 81.49

10 (23.3%) 33 (76.7%)

6 . 1 3 ~ 1.33 1 . 4 7 ~ 0.38 4 . 2 5 ~ 1.17 1 . 5 6 ~ 0.75

26.84 61.43 2 . 9 5 ~ 5.43

49.63 2 91.92

25 (46.3%) 29 (53.7%)

0.21 0.85 0.16 0.53

0.1 1 0.09 0.08 0.03

connecting the genotype with atherosclerosis was 2.79 (950/, CI 1.07-7.25). Small group sizes precluded the analysis of the interaction of apoA-I/C-I11 genotypes with apoB genotypes and apoE phenotypes.

We also clarified whether the apoE phenotype plays any major role in the recovery of the patients to a working ability on the average within 11 months from the stroke. Of a total of 215 subjects with a full working ability at the time of the brain attack, 40 (29 YO) out of the 139 subjects with the phenotypes E2/E3 or E3/E3 and 19 (25%) out of the 76 subjects with the phenotypes E3/E4 or E4/E4 were on sick leave or retired at follow-up, suggesting that the apoE phenotype has no major role in the prognosis of stroke patients in this respect.

Although a matter of debate, the insertion/deletion polymorphism of the ACE gene has been suggested to be associated with an increased risk of myocardial infarction (43-45). In the present study no differences in the ACE genotype distributions or allele frequencies were noticed when the patients were classified according to the presence or absence of atherosclerosis (Table 5 ) , or according to their blood pressure status (data not shown).

Discussion

Although ischaemic stroke may be caused by a variety of pathophysiological mechanisms, brain infarction resulting from atherosclerosis has been proposed to be the major underlying condition responsible for acute occlusive cerebrovascular events (46-48). Thus, in search for putative genetic factors affecting the risk of

ISCHAEMIC BRAIN DISEASE 229

Table 1. Angiotensin-converting enzyme (ACE) genotype distributions and allele frequencies in patients with or without cerebrovascular atherosclerosis.

Genetic group Cerebrovascular atherosclerosis

No Yes n n (%) n (YO)

Patients

~

ACE genotype 18 (20 9) 26 (17 6) II 44

di 117 41 (47 7) 76 (51 4) dd 73 27 (31 4) 46 (31 1) total 234 86 (100) 148 (100)

ACE alleles Allele I (%) 0 44 0 45 0 43 Allele d (%) 0 56 0 55 0 57

stroke, the genes whose mutations have been found to promote development of atherosclerotic vascular changes appear to be probable candidates. Although there is ample evidence of relatively common gene mutations modifying the risk of coronary events (for review, see (12)), information on their role in cerebrovascular disease remains scanty. Even in the case of CHD, the focus of molecular genetic studies has been virtually restricted to regulatory genes of lipoprotein meta-bolism, in particular to those encoding apolipoproteins and lipoprotein receptors.

Our own study on 234 patients with ischaemic stroke or TIA revealed that the risk of cerebrovascular atherosclerosis was associated with a combined genotype of apoE and apoB: atherosclerosis was present in the majority of the patients carrying both the E4 and X2 alleles. In addition, the apoE polymorphism was also associated with variation in serum triglyceride and HDL-cholesterol levels, whereas there were no such associations between the apoB gene polymorphism and serum lipid or lipoprotein levels.

Some important pitfalls should be kept in mind in the interpretation of our results. Firstly, a control group of subjects without cerebrovascular disease confirmed by angiography could not be included for obvious ethical reasons. Thus, comparisons between the genotypes and the extent of the disease process could only be carried out on a within-group basis. Comparison of the frequencies of specific alleles and genotypes in the patients and a random population (Table 1) may result in falsely conservative estimates of the significance of the individual gene loci, as many subjects in the population sample may ultimately develop cerebrovascular disease. In addition, the controls serving for allele frequency comparisons were selected from published population studies and they were slightly younger than the patients. Secondly, lipid and DNA studies were conducted using samples taken


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230 AALTO-SETALA PALOMAKI . MIETT~NEN ET AL

approximately 1 year after the initial event. This necessarily implies a loss of fraction of the initial cohort. There may have been some over-representation of patients with previously high lipid levels responding to invitations to blood sampling, and alterations in dietary habits in response to the disease event may have blunted associations between the lipid-regulatory genes and the serum lipoprotein levels. Thirdly, the power provided by the size of the cohort may not be sufficient to reveal subtle but significant differences of allele frequencies in subjects with and without atherosclerosis. In general, these potential biases might have led to conservative conclusions and have by no means led to overestimation of the true associations.

The apoE allele frequencies in our cohort of stroke patients were not significantly different from those observed in a normal Finnish population (Table 1). Although the allele frequencies of apoE were not different in the patients with or without cerebrovascular atherosclerosis, the E3E4 phenotype was present more often (36%) in the patients with radiologically confirmed atherosclerosis than in those without it (17%; P = 0.03), suggesting that in occasional cases of ischaemic stroke apoE genetic variation may have played a role in the promotion of vascular lesions. The relationship of apoE polymorphism and cerebrovascular disorders has been examined in a number of recent studies. An increased prevalence of the ~4 allele was found among Spanish patients suffering from nonhaemorrhagic stroke (49), while another study conducted in France showed that the E2E3 phenotype was more frequent in patients with ischaemic stroke than in their controls (50). Recently de Andrade et a1 (51) reported that after careful consideration of the contribution of established risk factors, the apoE genotype E2/3 was significantly associated with carotid artery atherosclerosis as defined by B-mode ultrasonography. Apparent discrep- ancies in different case-control studies may arise from differences in the ages of the subjects under study. In fact, Kuusisto et a1 (52) reported that the apoE phenotype is no longer an important risk factor of stroke or CHD in the age group of about 70 years.

The apoE phenotypes have been been found to be associated with serum total and LDL-cholesterol levels in several studies carried out both in random populations and in patients with CHD (30-32, 53, 54). In our sample of patients with stroke a similar although statistically nonsignificant trend, ie elevated levels of LDL cholesterol in carriers of the apoE ~4 allele, was found (Table 2). In our study, the apoE genotypes were also associated with some other changes in lipid levels: total triglycerides were elevated and HDL cholesterol decreased in patients with the E2E3 phenotype as compared with those with the E3E3 and E3E4/E4E4 phenotypes. The allele ~2 has been shown to be associated with a decreased rate of

conversion of VLDL to LDL (30), which may partly explain the occurrence of elevated serum triglycerides in carriers of this allele. However, our study does not support a role for the allele ~2 as a gene promoting cerebrovascular atherosclerosis or stroke (Table 1).

The apoE ~4 allele has recently received considerable attention because of its association with an increased risk of Alzheimer’s disease (for review, see ( 5 5 ) ) . Carriers of the ~4 allele have also been reported to show accelerated cognitive impairment with ageing (56) and increased deposition of amyloid [3-protein following head injury (57). In addition, cell culture studies have shown that apoE4, in contrast to apoE3, does not support neurite extension (58), which may be a detrimental characteristic following injury of the nervous tissue. These findings prompted us to clarify whether the apoE phenotype was related to the outcome of the patients in terms of their working ability 1 year after the attack. We found no such relation but it should be emphasized that all of the subjects of the initial patient cohort could not be examined.

The XbaI polymorphism of the apoB gene was not associated with the extent of cerebrovascular atherosclerosis, nor did the frequencies of the X1 and X2 alleles in our total patient cohort differ from those in the random Finnish population (Table 1). Monsalva and co-workers (59) found the X1 allele to associate with carotid and/or coronary artery disease in patients living in the London area. There appears to be contradictory information on the relative frequencies of the X1 and X2 alleles in survivors of myocardial infarction vs healthy controls (29, 60, 61). Our previous study on Finnish subjects with angiographically verified normal coronary arteries and atherosclerotic coronary vessels revealed no statistically significant association between the apoB XbaI alleles and the presence of coronary disease, although there was a trend favouring over- representation of the X I allele in patients with vascular changes (53). Although the X2 allele has been found to be associated with elevations of serum cholesterol and triglyceride levels in many populations by mechanisms still to be explored, no major support for the role of this allele as an atherosclerosis gene has been gathered until now. It is therefore of interest that in the present study a significant association with atherosclerosis was observed when the patients with the X2 allele of the apoB gene and the E4 allele of apoE were combined and compared with those without either of these alleles.

Our study failed to show any significant association between cervical atherosclerosis and the SstI polymorphism of the apoA-I/C-111 gene complex, another DNA variation proposed to serve as a genetic marker of primary hypertriglyceridaemia ( 17-22), premature CHD (23, 24) and ischaemic cerebro-


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GENETIC RISK FACTORS AND ISCHAEMIC BRAIN DISEASE 23 1

vascular disease (62-64). The mechanism by which this DNA polymorphism could affect serum lipid levels and propensity to atherosclerosis remains poorly understood. Studies with apoC-111 transgenic (15) and knock-out (16) animals have indicated that apoC-111 is an important regulator of triglyceride metabolism, and human studies have shown a relation between plasma apoC-111 and triglyceride levels (65, 66). Elevated serum triglyceride levels, alone or in combination with a low serum HDL concentration, have in turn been shown to be associated with the risk of coronary athero-sclerosis (67) and cerebrovascular disease (68, 69).

Most studies (43-45) have suggested an association between myocardial infarction and ACE gene polymorphism, with carriers of the deletion allele showing a higher disease risk. In our previous study no relationship was found between the ACE gene polymorphism and myocardial infarction occurring at a very young (< 45 years) age (70). Results from studies on essential hypertension have yielded even more contradictory data, with some demonstrating a relation between the ACE gene polymorphism and elevated blood pressure (35 , 71,72) but others failing to reveal such a relationship (73-77). In the present study the insertion/deletion polymorphism of the ACE gene did not associate with the extent of cervical atherosclerosis nor with the occurrence of hyper-, tension in patients with ischaemic stroke. Our data

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In conclusion, our data indicate that the frequencies of common allelic variants of a number of putative atherosclerosis genes, ie those encoding apolipoproteins A-UC-111, B and E, occur in similar frequencies in patients with ischaemic cerebrovascular disease and in a random population sample. We found evidence for an increased risk of atherosclerotic changes in carotid arteries in patients carrying both the apoB X2 allele and apoE ~4 allele, but the clinical significance of this association remains to be explored.

We thank Ms Kaija Kettunen for skilled technical assistance. This work was supported by research grants from the Medical Council of the Academy of Finland, the Sigrid Juselius Foundation, the Paavo Nurmi Foundation, the Paulo Foundation, the Finnish Heart Foundation and the University of Helsinki.

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Genetic risk factors and ischaemic cerebrovascular disease: role of common variation of the genes encoding apolipoproteins and angiotensin-converting enzyme

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