Association of Apolipoprotein E ε2 With White Matter Disease but Not With Microbleeds
Background and Purpose— Apolipoprotein E (apoE) alleles (ε2 and ε4) are associated with cerebral amyloid angiopathy, in which white matter disease and microbleeds are prominent features. The role of apoE in patients with microbleeds or white matter disease but no evidence of cerebral amyloid angiopathy has not been elucidated. We studied apoE alleles in relation to white matter disease and microbleeds in patients with transient ischemic attack or ischemic stroke.
Methods— We obtained brain MRI scans and apoE genotypes in 334 transient ischemic attack or ischemic stroke patients. Microbleeds were scored on a gradient echo MRI and white matter disease was examined on fluid attenuated inversion recovery MRI using a semiquantitative rating scale.
Results— Patients with moderate to severe white matter disease more frequently carried apoE ε2 alleles (25.2% versus 11.3%, P=0.001), but not apoE ε4 (26.6% in apoE ε4 carriers versus 25.9%; P=0.98). Adjustment for traditional risk factors did not modify this relationship (odds ratio, 2.9; 95% confidence interval, 1.5 to 5.3). There was no association between the presence of microbleeds and the apoE ε4 or apoE ε2 alleles.
Conclusions— ApoE alleles do not exert a major influence on the development of microbleeds, but apoE ε2 may be associated with development of moderate to severe white matter disease in transient ischemic attack and stroke patients.
Cerebral amyloid angiopathy with intracerebral hemorrhage is associated with the presence of microbleeds (MB), white matter disease (WMD), and the apolipoprotein (apoE) ε2 and apoE ε4 polymorphisms.1,2 Microbleeds and white matter disease are also commonly observed on MRI (gradient echo imaging [GRE]) of normal elderly individuals and in patients with transient ischemic attack or ischemic stroke.3,4 MB probably represent hemosiderin laden macrophages produced by microhemorrhages.5 Low cholesterol has been proposed as a risk factor for the development of MB and symptomatic intracerebral hemorrhage.6,7 ApoE genotypes influence cholesterol levels; apoE ε4 leads to higher cholesterol levels, and apoE ε2 leads to lower cholesterol levels.8 It has been hypothesized that the presence of MB might predict the occurrence of cerebral hemorrhagic complications in patients undergoing stroke thrombolysis or in patients treated with oral anticoagulants.9 A recent study indicated that patients with MB have more severe cognitive impairment.10
Given the role of apoE in the pathogenesis of cerebral amyloid angiopathy, we studied whether sporadic MB and WMD were related to the presence of apoE polymorphisms in transient ischemic attack or ischemic stroke patients.
Patients were selected from an ongoing prospective hospital based cohort study on the long term risks of microbleeds in white patients 50 years and older with cerebral ischemia (ischemic stroke and transient ischemic attack). Patients who were admitted to the Stroke Unit of the University Hospitals in Leuven, Belgium, between July 2003 and May 2005, and who survived at least 7 days after stroke were included. Patients with a history of intracerebral hemorrhage were excluded to obtain a homogenous population of ischemic stroke patients. Patients underwent both apoE genotyping and MRI. We recorded age at admission, sex, hypertension (defined as use of antihypertensive medication or physician measured blood pressure >140/90 mm Hg before stroke onset), diabetes mellitus (fasting glucose >125 mg/dL or use of antidiabetic medication), fasting cholesterol levels, and current smoking of the patients. Glucose levels were obtained after stroke. In patients with elevated fasting glucose levels, glycosylated hemoglobin measurements were obtained and an endocrinologist was consulted to make a final diagnosis of diabetes mellitus. A history of stroke was noted. Coronary artery disease was defined as history of myocardial infarction, coronary artery bypass grafting, coronary angioplasty, or stenting or angina pectoris.
ApoE genotypes were determined using the LightCycler-ApoE Mutation Detection Kit (Roche). Genotypers were blinded to imaging results.
All patients underwent an acute stroke imaging protocol including sagittal T1-weighted, axial T2-weighted, fluid-attenuated inversion-recovery imaging plus diffusion-weighted imaging and GRE. MRI scans were performed on a Siemens Magnetom Expert with a field strength of 1 Tesla or a Siemens Symphony Vision 1.5 Tesla system or a Philips Intera 1.5 or 3 Tesla system. The GRE sequences were as follows: repetition time (TR), 1000 ms; echo time (TE), 35 ms (Siemens Magnetom Expert), TR, 710 ms; TE, 26ms (Siemens Vision), and TR, 917 ms; TE, 16 ms (Philips Intera). The GRE imaging was obtained in the axial plane with the following parameters: 7-mm slice thickness, field of view 240 mm, 60° flip angle, and 256×256 matrix. Choice of scan type (1, 1.5, or 3 T) was determined by availability of the machine.
Fluid-attenuated inversion-recovery images were analyzed using a modified semiquantitative rating scale devised by Fazekas et al.11 This method yields 2 separate brain WMD scores: (1) subcortical and deep white matter lesions and (2) periventricular lesions. Each variable was scored on a scale of increasing severity. White matter lesions were scored by the following: 0, normal; 1, punctate; 2, coalescing; and 3, confluent. Periventricular lesions were scored by the following: 0, normal, pencil lines and/or caps, smooth haloes; and 1, irregular. For statistical analyses, WMD severity, defined as the sum of the scores on the white matter lesions and periventricular lesions rating scale, was dichotomized into scores 0 or 1 (absent or mild) versus ≥2 (moderate to severe).
MB were defined as homogeneous round hypointense lesions of diameter ≤5 mm on GRE. Hypointense lesions within the subarachnoid space and areas of symmetric hypointensity in the globus pallidus on GRE were considered to represent adjacent pial blood vessels or calcifications and were excluded. The number and location of MB were assessed independently by two trained observers (V.T., R.L., or M.S.). The locations of the MB were classified by cerebral region as follows: cortico-subcortical and white matter, basal ganglia, thalamus, and infratentorial area (brain stem, cerebellum). Observers where blinded to the results of apoE status.
Hardy Weinberg equilibrium for the apoE allele distribution was determined using GENEPOP in controls and in cases (MB or moderate to severe WMD).12 Differences in genotype and allele frequencies between groups were compared using the χ2 test; reported probability values are 2-sided. The level of significance was set at P<0.05. Adjusted analyses were performed using logistic regression with either the presence of MB or the presence of WMD as the dependent variable. All statistical tests were performed using SPSS 10.0. The study was approved by the Ethics Committee of the UZ Leuven and patients or their relatives provided informed consent.
Between July 2003 and May 2005, 783 patients 50 years or older with transient ischemic attack or ischemic stroke were admitted. Three hundred forty-two patients (44%) participated in the study. The 441 excluded patients were on average 4 years older (mean, 75±10 versus 71±10 years; P=0.001) and more frequently female (48% versus 38%; P=0.01) than the 342 participants. Among the participants there were 261 patients with stroke (76.3%) and 81 patients with transient ischemic attack (23.7%). The mean age was 71 years (standard deviation, 10 years) and 131 (38%) were female. A stroke history was present in 41 patients (12%). The baseline risk factors of the included patients are shown in Table 1.
MRI scans were obtained at 1 T in 99 patients (29%), at 1.5 T in 167 (49%), or at 3 T in 76 (22%) patients. The interobserver reliability for the presence of MB was substantial (κ=0.71; 95% confidence interval [CI], 0.59 to 0.82). The rate of detection of MB or WMD did not differ by scan type.
Eighty-nine patients (26%) had MB. Of these, 49 patients (55%) had at least 2 MB. The median number of MB in these patients was 2 (25th percentile to 75th percentile, 1 to 4). MB were present in cortico-subcortical regions in 81% of patients, in the basal ganglia in 12.4%, in the thalamus in 17%, in the brain stem in 15%, and in the cerebellum in 26%. MB were present in multiple locations in 31 patients (34.8%). In univariate analysis, only age was a risk factor for the presence of MB (Table 1). Low total cholesterol or low-density lipoprotein levels were not more frequently found in patients with MB. MB were strongly associated with WMD.
WMD was found in 273 (79.8%) patients. Mild WMD (modified Fazekas score of 1) was found in 134 (39.2%) patients and moderate to severe WMD (modified Fazekas score of 2, 3, or 4) was found in 139 patients (40.6%). The only significant clinical risk factors for moderate to severe WMD in univariate analysis were age and stroke history. MB were strongly associated with WMD. (Table 2)
The allele distributions of apoE were in Hardy Weinberg equilibrium (P=0.10 for cases; P=0.49 for controls [MB analyses]; P=0.94 for cases; P=0.83 for controls [WMD]). Patients with MB were not more frequently carriers of apoE ε4 alleles (n=22, 24.7% versus n=68, 26.9%; P=0.80) or apoE ε2 alleles (n=15, 16.9% versus n=43, 17%; P=1.00) (Table 3). In patients with MB, the presence of apoE ε4 did not lead to an increased number of MB (median number 2 in both groups; P=0.74). Similarly, the median number of MB was not increased in the presence of apoE ε2 (median 2 versus 3; P=0.09). Adjusted analyses with correction for age, sex, arterial hypertension, diabetes mellitus, stroke history, coronary artery disease, current smoking, and cholesterol levels did not change the results. Subgroup analysis of the 72 patients with corticosubcortical MB showed no association with apoE ε2 or apoE ε4.
Patients with moderate to severe WMD more frequently carried apoE ε2 alleles (n=35, 25.2% versus n=23, 11.3%; odds ratio, 2.63; 95% CI, 1.48 to 4.7), but not apoE ε4 (n=36, 25.9% in apoE ε4 carriers versus n=54, 26.6%; odds ratio, 0.96; 95% CI, 0.59 to 1.6).
After adjustment for age, sex, hypertension, diabetes mellitus, stroke history, coronary artery disease, current smoking, and fasting cholesterol levels and scan type, apoE ε2 was independently associated with the presence of WMD (adjusted odds ratio, 2.85; 95% CI, 1.53 to 5.31) (Table 4). ApoE ε4 was not associated with WMD in adjusted analyses (odds ratio, 0.96; 95% CI, 0.57 to 1.63).
ApoE ε2 carriers had lower cholesterol and low-density lipoprotein levels compared with patients without apoE ε2 (mean difference for cholesterol, 14 mg/dL; 95% CI, 3 to 26; for LDL, 17 mg/dL; 95% CI, 5 to 29).
Our results show that apoE ε2 may be associated with the development of WMD. Although we cannot exclude a type I error given the multiple tests that were performed, the relatively small sample size and the hospital-based design, we believe this may well be a genuine association. The association we found is strong, independent of multiple other risk factors, and replicates results from a population-based study in a white population.13 In the Austrian Stroke Prevention study, normal subjects with severe WMD on MRI more frequently carried an apoE ε2 allele, despite more favorable cholesterol levels and less severe cardiac disease.13 One case control study found a higher prevalence of apoE ε2 carriers in ischemic stroke patients and several studies report an association with intracerebral hemorrhage.14,15
ApoE alleles seem unlikely to influence the development of MB. Three previous reports have examined the role of apoE in the development of cerebral MB. In the Framingham study, apoE ε4 was not a risk factor for the presence of MB. The power of this study was limited as only 22 patients with MB were included.16 A larger study in an Asian population found a higher frequency of either apoE ε2 or ε4 in patients with lobar MB, consistent with the location of hemorrhage in patients with amyloid angiopathy, and with the location of MB in patients with hereditary cerebral hemorrhage of Dutch type.17 This finding is nevertheless difficult to interpret because neither the apoE ε2 or ε4 alleles were individually associated with the presence of MB. In a study from Austria, there was no relationship between the presence of MB and apoE in 101 patients with intracerebral hemorrhage.18
In epidemiologic studies, low cholesterol levels were found to be a risk factor for symptomatic intracerebral hemorrhage, although this is controversial.7 One previous study from Korea found lower cholesterol levels in patients with MB.6 We could not confirm this association. This may be related to differences in the genetic background because all our patients were white, or because of differences in study design. Further studies are needed to address this issue.
It is unclear how apoE ε2 might predispose to small vessel disease. A mechanism involving both the apoE ε2 and apoE ε4 allele has been proposed in relation to lobar intracerebral hemorrhage. ApoE ε2 could lead to changes in vessels loaded with amyloid, resulting in vascular ruptures.2 After ischemic insults direct atherogenic effects on cerebral microvasculature as well as defective repair mechanisms have been proposed.13 Another hypothesis arises from findings in cerebral amyloid angiopathy, in which apoE ε2 has been reported to be associated with fibrinoid necrosis.19
Previous studies have mainly focused on apoE ε4 and its relationship with ischemic stroke. A recent meta-analysis found no relationship between apoE ε4 and ischemic stroke.15 The relationship between apoE ε4 and WMD was examined in 2 prospective population-based studies. In the Rotterdam study apoE ε4 carriers were at increased risk for WMD, but only if they also had hypertension.20 The authors hypothesize that this may reflect a diminished capacity for neuronal repair in apoE ε4 carriers. This is supported by findings in the NHLBI Twin Study, in which patients with apoE ε4 and concomitant vascular diseases had more severe WMD and atrophy.21 We did not find a relationship between apoE ε4 and WMD, nor could we show an interaction between apoE ε4, hypertension, and the presence of WMD.
Our study has some limitations. Patient data were obtained in one hospital only and therefore could be biased. This was not a consecutive series of patients: included patients were younger, more frequently male, and survived at least 7 days after the index stroke. We did not include healthy controls as this was a stroke patient-based study. Consequently, overmatching could have obscured relationships between hypertension and WMD and MB. Furthermore, our sample size was relatively small and therefore not powered to detect small differences. The relative scarcity of rare apoE genotypes (apoE ε2/ε2) in our sample explains why we did not perform analysis by genotype. The relationship we found between WMD and apoE ε2 alleles was based on a crude scale. Future studies can be performed using quantitative MRI to detect pathologic processes in white matter and their relationship with apoE alleles, as recently shown in a study using healthy individuals.22
In conclusion, we report an association between apoE ε2 and WMD in stroke patients. The pathogenic mechanism leading to white matter injury in which the ε2 allele is involved needs to be elucidated. Bearing in mind these diverse findings concerning apoE, MB, and WMD, the question for a genome wide scan arises to find modifying genes in relationship to MB and WMD.23 Such findings could lead to new insights in the pathogenesis of stroke and people at risk.
- Received October 5, 2006.
- Revision received November 9, 2006.
- Accepted November 17, 2006.
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