Published online before print February 22, 2007, doi: 10.1161/01.STR.0000259816.31370.44
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
(Stroke. 2007;38:1185.)
© 2007 American Heart Association, Inc.
Original Contributions |
Association of Apolipoprotein E 
2 With White Matter Disease but Not With Microbleeds
From Department of Neurology, University Hospitals Leuven, Leuven, Belgium.
Correspondence to Vincent Thijs, MD, PhD, Department of Neurology, University Hospitals Gasthuisberg, Herestraat 49, B-3000 Leuven, Belgium. E-mail vincent.thijs{at}uz.kuleuven.ac.be
 |
Abstract |
|---|
 Top Abstract IntroductionMethodsResultsDiscussionReferences |
|---|
Background and Purpose— Apolipoprotein E (apoE) alleles (
2 and 4) 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 2 alleles (25.2% versus 11.3%, P=0.001), but not apoE 4 (26.6% in apoE 4 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 4 or apoE 2 alleles.
Conclusions— ApoE alleles do not exert a major influence on the development of microbleeds, but apoE 2 may be associated with development of moderate to severe white matter disease in transient ischemic attack and stroke patients.
Key Words: apolipoprotein E • genetics • white matter
|
Introduction |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Cerebral amyloid angiopathy with intracerebral hemorrhage is associated with the presence of microbleeds (MB), white matter disease (WMD), and the apolipoprotein (apoE)
2 and apoE 4 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 4 leads to higher cholesterol levels, and apoE 2 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.
|
Methods |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
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 Genotyping
ApoE genotypes were determined using the LightCycler-ApoE Mutation Detection Kit (Roche). Genotypers were blinded to imaging results.
MRI
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 256x256 matrix. Choice of scan type (1, 1.5, or 3 T) was determined by availability of the machine.
WMD
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).
Cerebral MB
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.
Statistics
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 
2 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.
|
Results |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Study Population
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.
|
Imaging Results
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 (
=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.
MB
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
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)
|
ApoE Alleles
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 4 alleles (n=22, 24.7% versus n=68, 26.9%; P=0.80) or apoE 2 alleles (n=15, 16.9% versus n=43, 17%; P=1.00) (Table 3). In patients with MB, the presence of apoE 4 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 2 (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 2 or apoE 4.
|
Patients with moderate to severe WMD more frequently carried apoE 2 alleles (n=35, 25.2% versus n=23, 11.3%; odds ratio, 2.63; 95% CI, 1.48 to 4.7), but not apoE 4 (n=36, 25.9% in apoE 4 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 2 was independently associated with the presence of WMD (adjusted odds ratio, 2.85; 95% CI, 1.53 to 5.31) (Table 4). ApoE 4 was not associated with WMD in adjusted analyses (odds ratio, 0.96; 95% CI, 0.57 to 1.63).
|
ApoE 2 carriers had lower cholesterol and low-density lipoprotein levels compared with patients without apoE 2 (mean difference for cholesterol, 14 mg/dL; 95% CI, 3 to 26; for LDL, 17 mg/dL; 95% CI, 5 to 29).
|
Discussion |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Our results show that apoE
2 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 2 allele, despite more favorable cholesterol levels and less severe cardiac disease.13 One case control study found a higher prevalence of apoE 2 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 4 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 2 or 4 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 2 or 4 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 2 might predispose to small vessel disease. A mechanism involving both the apoE 2 and apoE 4 allele has been proposed in relation to lobar intracerebral hemorrhage. ApoE 2 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 2 has been reported to be associated with fibrinoid necrosis.19
Previous studies have mainly focused on apoE 4 and its relationship with ischemic stroke. A recent meta-analysis found no relationship between apoE 4 and ischemic stroke.15 The relationship between apoE 4 and WMD was examined in 2 prospective population-based studies. In the Rotterdam study apoE 4 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 4 carriers. This is supported by findings in the NHLBI Twin Study, in which patients with apoE 4 and concomitant vascular diseases had more severe WMD and atrophy.21 We did not find a relationship between apoE 4 and WMD, nor could we show an interaction between apoE 4, 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 2/2) in our sample explains why we did not perform analysis by genotype. The relationship we found between WMD and apoE 2 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 2 and WMD in stroke patients. The pathogenic mechanism leading to white matter injury in which the 2 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.
|
Acknowledgments |
|---|
Disclosures
None.
Received October 5, 2006; revision received November 9, 2006; accepted November 17, 2006.
|
References |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
1. Greenberg SM, Briggs ME, Hyman BT, Kokoris GJ, Takis C, Kanter DS, Kase CS, Pessin MS. Apolipoprotein e epsilon 4 is associated with the presence and earlier onset of hemorrhage in cerebral amyloid angiopathy. Stroke. 1996; 27: 1333–1337.
2. Greenberg SM, Vonsattel JP, Segal AZ, Chiu RI, Clatworthy AE, Liao A, Hyman BT, Rebeck GW. Association of apolipoprotein e epsilon2 and vasculopathy in cerebral amyloid angiopathy. Neurology. 1998; 50: 961–965.
3. Roob G, Schmidt R, Kapeller P, Lechner A, Hartung HP, Fazekas F. MRI evidence of past cerebral microbleeds in a healthy elderly population. Neurology. 1999; 52: 991–994.
4. Koennecke HC. Cerebral microbleeds on MRI: Prevalence, associations, and potential clinical implications. Neurology. 2006; 66: 165–171.
5. Fazekas F, Kleinert R, Roob G, Kleinert G, Kapeller P, Schmidt R, Hartung HP. Histopathologic analysis of foci of signal loss on gradient-echo T2*-weighted MR images in patients with spontaneous intracerebral hemorrhage: evidence of microangiopathy-related microbleeds. AJNR Am J Neuroradiol. 1999; 20: 637–642.
6. Lee SH, Bae HJ, Yoon BW, Kim H, Kim DE, Roh JK. Low concentration of serum total cholesterol is associated with multifocal signal loss lesions on gradient-echo magnetic resonance imaging: analysis of risk factors for multifocal signal loss lesions. Stroke. 2002; 33: 2845–2849.
7. Yano K, Reed D, MacLean C. Serum cholesterol and hemorrhagic stroke in the Honolulu heart program. Stroke. 1989; 20: 1460–1465.
8. Sing CF, Davignon J. Role of the apolipoprotein E polymorphism in determining normal plasma lipid and lipoprotein variation. Am J Hum Genet. 1985; 37: 268–285.[Medline] [Order article via Infotrieve]
9. Fan YH, Zhang L, Lam WW, Mok VC, Wong KS. Cerebral microbleeds as a risk factor for subsequent intracerebral hemorrhages among patients with acute ischemic stroke. Stroke. 2003; 34: 2459–2462.
10. Werring DJ, Frazer DW, Coward LJ, Losseff NA, Watt H, Cipolotti L, Brown MM, Jager HR. Cognitive dysfunction in patients with cerebral microbleeds on T2*-weighted gradient-echo MRI. Brain. 2004; 127: 2265–2275.
11. Fazekas F, Chawluk JB, Alavi A, Hurtig HI, Zimmerman RA. MR signal abnormalities at 1.5 T in Alzheimer’s dementia and normal aging. AJR Am J Roentgenol. 1987; 149: 351–356.
12. Raymond M, Rousset F. Genepop (version-1.2)—population-genetics software for exact tests and ecumenicism. J Heredity. 1995; 86: 248–249.
13. Schmidt R, Schmidt H, Fazekas F, Schumacher M, Niederkorn K, Kapeller P, Weinrauch V, Kostner GM. Apolipoprotein E polymorphism and silent microangiopathy-related cerebral damage. Results of the Austrian Stroke Prevention study. Stroke. 1997; 28: 951–956.
14. Kokubo Y, Chowdhury AH, Date C, Yokoyama T, Sobue H, Tanaka H. Age-dependent association of apolipoprotein E genotypes with stroke subtypes in a Japanese rural population. Stroke. 2000; 31: 1299–1306.
15. Sudlow C, Martinez Gonzalez NA, Kim J, Clark C. Does apolipoprotein E genotype influence the risk of ischemic stroke, intracerebral hemorrhage, or subarachnoid hemorrhage? Systematic review and meta-analyses of 31 studies among 5961 cases and 17,965 controls. Stroke. 2006; 37: 364–370.
16. Jeerakathil T, Wolf PA, Beiser A, Hald JK, Au R, Kase CS, Massaro JM, DeCarli C. Cerebral microbleeds: prevalence and associations with cardiovascular risk factors in the Framingham study. Stroke. 2004; 35: 1831–1835.
17. Kim M, Bae HJ, Lee J, Kang L, Lee S, Kim S, Lee JE, Lee KM, Yoon BW, Kwon O, Koo JS, Kim BK. ApoE epsilon2/epsilon4 polymorphism and cerebral microbleeds on gradient-echo MRI. Neurology. 2005; 65: 1474–1475.
18. Seifert T, Lechner A, Flooh E, Schmidt H, Schmidt R, Fazekas F. Lack of association of lobar intracerebral hemorrhage with apolipoprotein E genotype in an unselected population. Cerebrovasc Dis. 2006; 21: 266–270.[CrossRef][Medline] [Order article via Infotrieve]
19. McCarron MO, Nicoll JA, Stewart J, Ironside JW, Mann DM, Love S, Graham DI, Dewar D. The apolipoprotein E epsilon2 allele and the pathological features in cerebral amyloid angiopathy-related hemorrhage. J Neuropathol Exp Neurol. 1999; 58: 711–718.[Medline] [Order article via Infotrieve]
20. de Leeuw FE, Richard F, de Groot JC, van Duijn CM, Hofman A, Van Gijn J, Breteler MM. Interaction between hypertension, apoE, and cerebral white matter lesions. Stroke. 2004; 35: 1057–1060.
21. DeCarli C, Reed T, Miller BL, Wolf PA, Swan GE, Carmelli D. Impact of apolipoprotein E epsilon4 and vascular disease on brain morphology in men from the NHLBI Twin Study. Stroke. 1999; 30: 1548–1553.
22. Persson J, Lind J, Larsson A, Ingvar M, Cruts M, Van Broeckhoven C, Adolfsson R, Nilsson LG, Nyberg L. Altered brain white matter integrity in healthy carriers of the apoE epsilon4 allele: a risk for AD. Neurology. 2006; 66: 1029–1033.
23. DeStefano AL, Atwood LD, Massaro JM, Heard-Costa N, Beiser A, Au R, Wolf PA, DeCarli C. Genome-wide scan for white matter hyperintensity: The Framingham heart study. Stroke. 2006; 37: 77–81.
This article has been cited by other articles:
 |
|
 |
|
  C. Cordonnier, G. M. Potter, C. A. Jackson, F. Doubal, S. Keir, C. L.M. Sudlow, J. M. Wardlaw, and R. A.-S. Salman Improving Interrater Agreement About Brain Microbleeds: Development of the Brain Observer MicroBleed Scale (BOMBS) Stroke, January 1, 2009; 40(1): 94 - 99. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 APOE Genotype and MRI Markers of Small Vessel Disease Journal Watch Neurology, June 5, 2007; 2007(605): 1 - 1. [Full Text]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||