Association of Apolipoprotein E4 and Haplotypes of the Apolipoprotein E Gene With Lobar Intracerebral Hemorrhage
Background and Purpose— Conflicting reports in the literature exist with regard to the association of apolipoprotein E (apo E) alleles and lobar intracerebral hemorrhage (ICH). We genotyped 12 single-nucleotide polymorphisms in the 5′ upstream regulatory, exonic, and intronic regions of the apo E gene and performed genotype and haplotype association analyses.
Methods— We prospectively enrolled subjects with hemorrhagic stroke and matched them with 2 controls based on age, race, and sex. Each case was reviewed by a physician to determine case status and location of the ICH. Multivariate logistic-regression modeling with backward elimination was used to determine significant risk factors for lobar ICH. Associations at the genotype and haplotype levels and linkage disequilibrium were conducted according to standard statistical methods.
Results— Between May 1997 and December 2002, 315 cases of ICH were recruited, of whom 107 were lobar ICH cases matched to 205 controls. No association was found for apo E2, E3, or E4 with nonlobar ICH. Independent, significant risk factors for lobar ICH included apo E4, untreated hypertension, anticoagulant use, a first-degree relative with ICH, and ≤high school education (compared with >high school education). Treated hypercholesterolemia compared with “no history of hypercholesterolemia” was associated with a decreased risk of lobar ICH. Haplotype association analysis demonstrated a significant association of the apo E gene with lobar ICH among whites (P<0.0001) and blacks (P=0.0024).
Conclusions— Apo E4 is independently associated with lobar ICH but not nonlobar ICH. Haplotypes of the apo E gene are associated with lobar ICH. Untreated hypertension is a risk factor for lobar ICH.
Spontaneous intracerebral hemorrhage (ICH) occurs at an annual incidence rate of 15 to 19 per 100 000.1 ICH has been shown to have important genetic and environmental risk factors, including apolipoprotein E (apo E) alleles and hypertension.2–7
Conflicting results regarding the association of the apo E type ε2 (apo E2) and/or ε4 (apo E4) allele with lobar ICH have been reported.2–6 Greenberg et al5,6 reported an association between apo E4 and lobar ICH, but Yamada et al2 were unable to confirm the association in a Japanese population. Nicoll et al4 did not find an association with apo E4 but reported that apo E2 was associated with lobar ICH. McCarron and Nicoll3 reported that apo E2 was specific for cerebral amyloid angiopathy (CAA)–related hemorrhage. These studies, however, did not examine the potential effect of regulatory regions or nearby mutations.
See Editorial Comment, pg 1879
In a previous study, we found that having either an apo E4 or an apo E2 allele was a risk factor for lobar ICH, but the study had insufficient power to determine which of the alleles led to the association.7 No report of other polymorphisms of the apo E gene and the risk of lobar ICH exists. The −491 AA promoter polymorphism was found to increase amyloid deposition among subjects with apo E4 but not among those without an apo E4 allele.8 Given the shared findings of cerebral amyloid angiopathy in lobar ICH and in Alzheimer’s disease, we included promoter and regulatory region polymorphisms of the apo E gene into our analysis.
Association studies of common variants have been proposed as powerful approaches for identifying genetic variants underlying complex diseases.9 A haplotype is defined as a combination of multiple alleles on 1 chromosome. Haplotype analysis refers to the simultaneous study of multiple alleles. Such studies are facilitated by linkage disequilibrium (LD) mapping, in which alleles at adjacent loci are inherited together because of their nonrandom gametic association. Thus, each single-nucleotide polymorphism (SNP) provides information on other mutations that are near the SNP. In addition, the effect of regulatory regions on disease-causing mutations may be observed. In this study, we performed haplotype analyses to assess the levels of association between SNPs on the apo E gene and lobar ICH.
The methods of the Genetic and Environmental Risk Factors of Hemorrhagic Stroke Study have been published previously.7,10,11 The study was approved by the institutional review boards of all participating hospitals. All patients with a potential ICH or subarachnoid hemorrhage and residing within 50 miles of the University of Cincinnati are identified by surveillance of hospital emergency and radiology departments and hospital discharge diagnoses.
ICH was defined as a nontraumatic, abrupt onset of severe headache, altered level of consciousness, and/or focal neurologic deficit associated with a focal collection of blood within the brain parenchyma not due to hemorrhagic conversion of a cerebral infarct. Patients were eligible if they were ≥18 years old and had no evidence of trauma or brain tumor as the cause of hemorrhage. Cases of vascular malformation related hemorrhage were excluded from the current analysis.
A subset of cases was approached for direct interview and genetic sampling. If the patient was unable to be interviewed, a proxy was interviewed. We recorded a history of hypertension as well as medications use. Medications were reviewed and classified as antihypertensive medication by a study physician (D.W.).10 Untreated hypertension was defined as a history of hypertension but not being treated with antihypertensive medication at the time of admission.
Two controls of the same sex, race, and age (±5 years) for each interviewed case were identified. Each subject was interviewed in a highly structured and identical manner. Four buccal brush samples were obtained from each subject.
Apo E SNPs
SNP selection was based on position and function, relative physical distance, and heterozygosity rates. Coding SNPs causing nonsynonymous (rs429358, rs7412, rs769452, and rs769455) and synonymous (rs157581 and rs1160983) changes and SNPs of promoter (rs769446 and rs405509) and 5′ untranslated (rs440446, rs10119, rs1160984, and rs1160985) regions were chosen from the NCBI SNP database. Apo E alleles were determined by genotypes at rs429358 and rs7412.12 Cytosines at both loci equal apo E4, thymines at both equal apo E2, and thymine at the first and cytosine at the second equal apo E3. Genotyping was performed with the TaqMan assay.
The data were manipulated and analyzed with SAS software (SAS Institute). Univariate analyses were performed with exact conditional logistic regression modeling, and multivariable analyses were performed with conditional logistic modeling. Backward elimination was used for risk factors with significance levels of P<0.10. Apo E2 and apo E4 were forced into the model. Analyses were performed for lobar and nonlobar ICH.
Case-control comparison was performed separately by race, with univariate comparisons performed by χ2. All SNPs were found to be in Hardy-Weinberg equilibrium except for rs1160983, which was dropped from the analysis. Haplotype construction, analysis, and LD determination were performed with PHASE and confirmed with EMLD, HAPLOSTATS, HAPLOVIEW, and HELIXTREE programs. SNPs were dropped from haplotype construction if their minor allele frequency was <5% (rs1160984, rs769452, and rs769455). Haplotypes were created and compared with the HELIXTREE program, with replication performed by the HAPLOVIEW program. Subhaplotypes were created with a D′ cutoff of 0.70 as well as the 2 alleles that determine apo E status. A global statistical test for haplotype analysis was performed with the R/C method.13 Multiple comparison correction was performed with the Bonferroni method.
Between May 1997 and December 2002, 315 cases of ICH were enrolled. Of these, 112 had a lobar ICH, of whom 5 had experienced a prior hemorrhage, leaving 107 cases. Of these, 86 were white; 19, black; 1, white Hispanic; and 1, Asian. Of these, 98 had 2 matching controls and 9 had 1 matching control.
None of the individual polymorphisms were associated with lobar ICH after controlling for multiple testing (see online supplement). LD was determined for each pair of polymorphisms for whites and blacks (Figures 1 and 2⇓). Normalized disequilibrium (D′ ranges from −1 to 1) is a measure of how closely 2 polymorphisms are observed to occur together. A D′ close to or equal to 1 suggests that the allele at 1 locus predicts the allele at the other.
Table 1 presents the prevalence and univariate and multivariate risks for lobar ICH. The rs769455 locus was found to be significant in multivariate analysis. However, the heterozygous genotype appeared in only 1% of controls and in 4% of cases, and the minor homozygous genotype was not seen. Thus, it was dropped from the final multivariate model. In our population, the presence of apo E4 was independently associated with lobar ICH. Apo E2 was not a significant risk factor for lobar ICH in our study.
Genotyping was available for 111 cases and 214 controls, of whom 91 cases and 178 controls were white. The rs1160984, rs769452 and rs769455 SNPs were dropped because of a minor allele frequency <5%. The overall haplotype analysis (8 SNPs) between cases of lobar ICH and controls (Tables 2 and 3⇓) was significant for both whites (P<0.0001) and blacks (P=0.008) and remained so after correcting for multiple comparisons (P=0.005 for whites and P=0.03 for blacks). The most common haplotypes containing apo E2, E3, and E4 did not demonstrate significant associations with lobar ICH. Table 4 presents pairwise haplotype associations among whites and blacks. Additional haplotype analyses are available in the online supplement.
Controversial literature exists with regard to the association of apo E alleles and lobar ICH. We report that apo E4 is independently associated with lobar ICH. We support this finding with an overall haplotype association of the apo E gene with lobar ICH. Additional SNPs included as haplotypes also led to a haplotype association between the apo E gene and lobar ICH.
These differences may help explain some of the conflicting findings in the literature. Although we did not find an association between the apo E2 allele and lobar ICH, we did find that haplotypes that contained the apo E2 allele were associated with lobar ICH and that the most common haplotypes that contained apo E4 were not associated with lobar ICH. Thus, if the populations studied by Nicoll et al4 and McCarron and Nicoll3 contain a large proportion of these haplotypes, an association of apo E2 with lobar ICH could be found.
One hypothesis to explain these findings is that mutations of the regulatory regions may affect risk. Our study is the first report on the 5′ upstream regulatory and promoter regions of the apo E gene and the risk of ICH and is congruent with prior studies reporting that mutations of the promoter region may mediate the risk of Alzheimer disease.14–16
The question arises as to whether or not the haplotype association is being driven primarily by apo E4 or apo E3 alleles. If there were no additional risk or modifying risk by the other polymorphisms in the haplotype, then the risks accorded by apo E4 or apo E3 would be relatively uniform across haplotypes, and the most common haplotypes would be the most likely to demonstrate the effect, given a higher power to detect an association. However, we have found that the most common haplotypes had no association with risk, which reinforces the possibility that other polymorphisms may affect risk. This hypothesis requires confirmation through biologic studies of the relation of mutations in these regions with apo E production and/or function.
The biologic mechanism of the association of apo E with lobar ICH may be explained through their association with CAA. CAA occurs in 50% to 79% of patients with Alzheimer disease, which has also been associated with apo E4.17,18 In addition to the studies described earlier, McCarron and Nicoll3 reported that the risk of apo E2 with CAA occurred among subjects with and without Alzheimer disease, whereas the association of apo E4 with CAA was correlated with concomitant Alzheimer disease. This suggests that apo E2 is a specific risk factor for CAA-related hemorrhage, whereas apo E4 is related to CAA in general.
The amyloid precursor protein is cleaved by α-secretase, leaving a transmembrane portion of the protein ranging in length from 37 to 42 amino acids. Researchers have found that β-amyloid-42 is significantly elevated in Alzheimer disease patients and their first-degree relatives.19 McCarron et al20 reported that patients with CAA-related hemorrhage were more reactive to β-amyloid-42. Rosand et al21 reported an association of apo E2 among 41 patients with warfarin-related ICH compared with 66 controls, in which 7 of 11 subjects had pathologic evidence of CAA. In animal models, knockin mice with human apo E4 developed amyloid plaques as well as CAA, whereas apo E3 knockin mice developed almost no CAA or parenchymal plaques.22
We were surprised to find that untreated hypertension was a significant risk factor for lobar ICH. Previously, we reported that hypertension appeared to be a significant risk factor for nonlobar ICH but not lobar ICH.7 Later, we reported that untreated hypertension was a greater risk factor than treated hypertension for ICH.10 With double the sample size of our initial report, we included treated and untreated hypertension into the analysis and found that untreated hypertension was a significant risk factor for lobar ICH.
A limitation of our analysis is that survival bias may have affected our results. However, we have reported that our interviewed cases were similar to noninterviewed cases with respect to major risk factors.10,11 In addition, interrater agreement on assessment of the location of ICH was not evaluated and may have led to misclassification bias. Multiple testing is always a limitation in genetic association studies, particularly with haplotype analyses. Finally, the haplotype analysis was not adjusted for the presence of other factors, because individual haplotype assignment would include additional assumptions that may be less reliable. Independent confirmation of our findings in a different cohort is required.
Our study confirms and extends the literature supporting an association of apo E4 with lobar ICH. It further demonstrates that inclusion of polymorphisms of the 5′ upstream and promoter into haplotypes yields a significant association with lobar ICH, suggesting that other mutations in the region may affect risk.
This study was funded by grants from the National Institute of Neurological Diseases and Stroke (R-01-NS 36695) and the National Institute of Environmental Health (R-01-ES 06096), National Institutes of Health, Bethesda, Md.
Presented as the Robert G. Siekert New Investigator Award in Stroke at the 29th International Stroke Conference, San Diego, CA, February 6, 2004.
- Received April 18, 2005.
- Accepted May 13, 2005.
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