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Association between Genetic Polymorphisms and Sites of Cervicocerebral Artery Atherosclerosis
Association between Genetic Polymorphisms and Sites of Cervicocerebral Artery Atherosclerosis Aurauma Chutinet, MD,* Nijasri C. Suwanwela, MD,* Thiti Snabboon, MD,† Napasri Chaisinanunkul, MD,* Karen L. Furie, MD, MPH,‡ and Kammant Phanthumchinda, MD*
Ischemic stroke is a multifactorial disease with strong genetic elements. The purpose of this case-control study was to find relationships between apolipoprotein E (APOE), angiotensin-converting enzyme (ACE), and methylenetetrahydrofolate reductase (MTHFR) genotypes and atherosclerosis of the extracranial internal carotid and intracranial arteries in the Thai population. Patients aged .45 years with significant intracranial stenosis (IC group) or extracranial carotid artery stenosis (EC group) diagnosed by duplex ultrasound and/or computed tomography angiography (CTA) or magnetic resonance angiography (MRA) were studied. The control group comprised volunteers with no history of stroke and no evidence of significant cervicocerebral artery stenosis by ultrasound. Genomic DNA was extracted and genotyped for APOE isoforms, ACE insertion/deletion (I/D) polymorphism, and MTHFR C677T polymorphisms. There were 141 cases (83 in the IC group and 58 in the EC group) and 167 controls. The APOE e3/e4 genotype and APOE e4 allele were significantly associated with extracranial carotid artery stenosis (odds ratio, 2.55; 95% confidence interval, 1.07-6.05 and odds ratio, 2.85; 95% confidence interval, 1.35-5.99, respectively). These associations were not observed in patients with intracranial atherosclerosis. There was no significant association between ACE and MTHFR polymorphisms and stenosis at any site. In a multivariate model, sex, diabetes mellitus, hypertension, ischemic heart disease, and APOE e4 allele remained predictive of extracranial atherosclerosis. In our Thai population, the e4 allele in the APOE gene contributes to the genetic susceptibility of extracranial internal carotid atherosclerosis. The low prevalence of extracranial carotid stenosis in this population might result from low frequencies of the APOE e4 allele. Key Words: Apolipoprotein E—angiotensin-converting enzyme—methylenetetrahydrofolate reductase—intracranial stenosis—extracranial carotid stenosis. Ó 2012 by National Stroke Association
From the *Division of Neurology; †Division of Endocrinology, Department of Medicine, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand; and ‡Department of Neurology, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts. Received April 29, 2010; accepted October 10, 2010. Supported by the Neurological Society of Thailand. Address correspondence to Aurauma Chutinet, MD, Division of Neurology, Department of Medicine, Faculty of Medicine, Chulalongkorn University, Rama IV Road, Bangkok 10330, Thailand. E-mail: [email protected]. 1052-3057/$ - see front matter Ó 2012 by National Stroke Association doi:10.1016/j.jstrokecerebrovasdis.2010.10.002
Ischemic stroke is the leading cause of death and longterm disability worldwide.1 Among the different subtypes, atherosclerosis accounts for at least one-third of the cases.2,3 The common sites of cervicocerebral artery atherosclerosis are known to differ by race and ethnicity; for example, intracranial disease is more common in Asians and African Americans, whereas extracranial carotid stenosis is more common in Caucasians.4-8 Genetics, along with environmental factors, may play important roles in determining the site of atherogenesis and progression. A recent meta-analysis examining the association of genetic polymorphisms and susceptibility to
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stroke in Asian populations found significant associations of the 3 leading genes—the apolipoprotein E (APOE), angiotensin-converting enzyme (ACE), and methylenetetrahydrofolate reductase (MTHFR) genes—with stroke.9-11 Moreover, the APOE e4 allele has been found to be associated with intima-media thickness and stenosis of the extracranial carotid artery.12-14 None of those studies attempted to compare gene polymorphisms among various sites of vascular stenosis related to stroke, however. We postulated that genetic factors might play a role in determining the site of atherosclerosis, and accordingly, compared polymorphisms of APOE, ACE, and MTHFR in patients with significant extracranial atherosclerosis, those with intracranial atherosclerosis, and controls.
Patients and Methods Study Population This case-control study included Thai patients with significant ($50%) extracranial internal carotid and/or ($50%) intracranial atherosclerosis aged .45 years seen at King Chulalongkorn Memorial Hospital between March 2008 and February 2009. The degree of stenosis was determined by transcranial Doppler ultrasonography (TCD) and carotid duplex ultrasound (CDUS) and either computed tomography angiography (CTA) or magnetic resonance angiography (MRA). Patients with nonatherosclerotic causes of vascular stenosis (eg, dissection, vasculitis, moyamoya disease) were excluded. After informed consent was provided, baseline data (including demographic data, relevant past medical history, and family history of stroke and ischemic heart disease) were obtained, and a physical examination was performed. Blood samples for routine blood chemistry and genetic analyses were collected prospectively. The control group comprised healthy interval age-matched Thai volunteers who were members of an elders club at King Chulalongkorn Memorial Hospital. All control subjects were screened by questionnaire and physical examination to exclude previous stroke. TCD and CDUS were performed in all controls to exclude significant intracranial and extracranial carotid stenosis. This study was approved by the local Ethics Committee from the Institutional Review Board of the Faculty of Medicine, Chulalongkorn University (COA no. 676/2008; IRB no. 299/51). Written informed consent was obtained from each participant.
Evaluation for Arterial Stenosis To identify arterial stenosis, all enrolled patients were examined by TCD (Multidopp T, DWL-Compumedics, Germany) and CDUS (LOGIQ5 Expert, GE Healthcare, USA). In 90% of cases, MRA or CTA was performed and evaluated by a neuroradiologist to confirm the diagnosis of arterial stenosis. The interpreters of the MRA/CTA
were blinded to the CDUS and TCD results. The TCD criteria used to define $50% arterial stenosis were based on peak systolic flow velocity, as follows: .140 cm/s for middle cerebral artery, .120 cm/s for the anterior cerebral and internal carotid arteries, and .100 cm/s for the posterior cerebral, basilar, and vertebral arteries.15 Significant ($50%) extracranial stenosis on CDUS was defined according to previously published criteria.16 Patients with $50% stenosis of the extracranial internal carotid artery according to the ultrasound criteria were designated the EC group, and those with $50% intracranial artery stenosis without significant extracranial stenosis were designated the IC group.
Risk Factor Evaluation To identify the risk factors for atherosclerosis, a detailed history of established risk factors and medical history were obtained from each patient and prospectively recorded in the case record form. Baseline data included age, sex, presence of hypertension, diabetes mellitus, coronary heart disease, dyslipidemia, cigarette smoking, alcohol drinking, and history of stroke or coronary heart disease. Blood samples were collected for complete blood count, fasting plasma glucose, total cholesterol, triglyceride, high-density lipoprotein, and low-density lipoprotein.
Genotyping Genomic DNA was extracted from peripheral blood leukocytes by standard procedures with a DNA Mini Kit (Qiagen, Hilden, Germany). Genotyping for APOE isoforms, ACE I/D polymorphism, and MTHFR C677T polymorphism were analyzed. For APOE genotyping, DNA was amplified using two primers: 5’-ACAGAA TTCGCCCCGGCCTGGTACACAC-3’ and 5’-TAAGCTT GGCACGGCTGAAGGA-3’. Each amplification reaction contained 1 mg of leukocyte DNA, 1 pmol/mL of each primer, 10% dimethyl sulfoxide, and 2 units of Taq polymerase in a final volume of 30 mL. Each reaction mixture was heated at 95 C for 5 minutes, followed by 40 cycles of 95 C for 60 seconds, 65 C for 80 seconds, and 72 C for 80 seconds, with a final extension at 72 C for 7 minutes. The PCR products were treated with ExoSAP-IT (USP, Cleveland, OH) according to the protocols supplied by the manufacturer, and sent for direct sequencing to the Macrogen, Seoul, South Korea. For ACE I/D polymorphism, we used two primers, 5’-CTGGAGACCACTCCCATCATTTCT-3’ and 5’-GATGTG GCCATCACATTGGTCA GAT-3’, to detect a 190-bp fragment in the absence of insertion and a 490-bp fragment in the presence of insertion in the ACE gene. The amplicons were separated on 1% agarose gels and visualized using ethidium bromide staining and ultraviolet light. For MTHFR C677T, we used primers with the sequences 5’-ATCCCTATTGGC AGGTTAC-3’ and 5’-AGGACGGTGCGGTCAGAGTG-3’ in PCR reactions, and performed amplification using
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Table 1. Comparison of baseline characteristics and major risk factors in controls, the EC group, and the IC group Baseline characteristics and major risk factors Males, n (%) Age, years, mean 6 SD (95% CI) Diabetes, n (%) Hypertension, n (%) Ischemic heart disease, n (%) Dyslipidemia, n (%) Smoker, n (%) Alcohol drinking, n (%) Family history of ischemic heart disease, n (%)
Controls (n 5 167)
EC (n 5 58)
IC (n 5 83)
89 (53.3) 65.9 6 10.0 (64.4-67.4)
46 (79.3)* 72.4 6 8.9 (70.1-74.8)
53 (63.9) 65.2 6 10.5 (62.9-67.5)
13 (7.8) 92 (55.4) 10 (6.0) 151 (91.0) 10 (6.0) 24 (14.5) 12 (7.2)
27 (46.6)* 53 (91.4)* 15 (25.9)* 55 (94.8) 8 (14.5) 6 (10.9) 5 (9.1)
27 (33.3)* 71 (87.7)* 9 (11.1) 75 (92.6) 6 (7.5) 2 (2.5)* 6 (7.5)
*Statistically significant compared with controls.
initial denaturation at 94 C for 5 minutes, followed by 35 cycles of 94 C for 45 seconds, 60 C for 45 seconds, and 72 C for 45 seconds with a final extension at 72 C for 10 minutes. The C677T mutation creates a SchI recognition site, so digestion of the PCR product will generate two fragments (168 and 98 bp) that were size-fractionated on 1% agarose gels.
Statistical Analysis The risk factor profiles were compared between patients with significant stenosis of the extracranial carotid artery, those with significant stenosis of the intracranial arteries, and controls. Continuous variables were compared using an independent t test. Categorical variables were compared using the c2 test. Statistical significance was defined as a 5 0.05 by a two-tailed test. Logistic regression analysis was performed to identify the contributions of genetic polymorphisms and risk factors to the site of vascular stenosis.
Results Between March 2008 and February 2009, 308 subjects were included into the study (141 with significant stenosis
and 167 controls). Among the two groups of stenosis, 58 were in the EC group and 83 were in the IC group. Baseline characteristics are shown in Table 1. Males were predominant in all groups. The mean age was higher in the EC group. Diabetes mellitus, hypertension, ischemic heart disease, and smoking were significantly associated with stenosis. Male sex, diabetes mellitus, hypertension, and ischemic heart disease were significantly associated with extracranial carotid stenosis (P 5 .001, , .001, , .001, and , .001, respectively), whereas diabetes mellitus, hypertension, and alcohol consumption were significantly associated with intracranial artery stenosis (P , .001, , .001, and .003, respectively).
Genetic Study APOE Gene APOE genotypes and allele frequencies are shown in Table 2. The e3/e3 genotype was most prevalent in all groups and was present in 65%-75% of the cases. The e3/e4 genotype was more common in EC group (22.4%) compared with controls. The APOE e3 allele was the most common allele, found in .75% of subjects across
Table 2. Comparison of APOE genotypes in controls, the EC group, and the IC group EC (n 5 58)
IC (n 5 83)
APOE
Controls (n 5 167), n (%)
n (%)
OR (95% CI)
N (%)
OR (95% CI)
e2/e2 e3/e3 e4/e4 e2/e3 e2/e4 e3/e4 Allele e2 Allele e3 Allele e4
2 (1.2) 123 (73.7) 0 23 (13.8) 2 (1.2) 17 (10.2) 29 (8.7) 286 (85.6) 19 (5.7)
0 37 (67.3) 1 (1.7) 5 (8.6) 2 (3.4) 13 (22.4) 7 (6.0) 92 (79.3) 17 (14.6)
0 0.63 (0.32-1.25) 0 0.59 (0.16-1.75) 2.95 (0.29-30.10) 2.55 (1.07-6.05)* 0.68 (0.26-1.67) 0.64 (0.36-1.15) 2.85 (1.35-5.99)*
1 (1.2) 63 (75.9) 1 (1.2) 9 (10.8) 1 (1.2) 8 (9.6) 12 (7.2) 143 (86.1) 11 (6.6)
1.01 (0.20-5.02) 1.13 (0.59-2.17) 0 0.76 (0.31-1.84) 1.01 (0.20-5.02) 0.94 (0.35-2.44) 0.82 (0.38-1.73) 1.04 (0.59-1.85) 1.18 (0.51-2.68)
*Statistically significant compared with controls.
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Table 3. Multiple logistic regression analysis between controls and the EC group Risk factor
Crude OR (95% CI)
Adjusted OR (95% CI)
Female sex Age Diabetes mellitus Hypertension Ischemic heart disease Smoker APOE e3/e4 APOE e4 allele
0.3 (0.15-0.60)* 4.7 (1.76-12.35)* 10.2 (4.76-22.05)* 8.5 (3.24-22.42)* 5.4 (2.28-12.97)* 2.6 (0.99-7.11) 2.5 (1.07-6.05)* 2.8 (1.35-5.99)*
0.3 (0.15-0.82)* 3.2 (0.95-10.58) 5.6 (2.32-13.44)* 5.1 (1.76-14.81)* 3.0 (1.04-8.87)* – – 2.8 (1.10-7.05)*
*Statistically significant.
all groups. APOE e3/e4 genotype and the e4 allele were significantly associated with extracranial carotid stenosis compared with controls (odds ratio [OR], 2.55; 95% confidence interval [CI], 1.07-6.05 vs OR, 2.85; 95% CI, 1.35-5.99) (Table 2). Logistic regression analysis was performed to determine the predictors of extracranial carotid stenosis. On multivariate regression, sex, diabetes mellitus, hypertension, ischemic heart disease, and APOE e4 allele were independently associated with extracranial carotid stenosis (APOE e4 allele: OR, 2.8; 95% CI, 1.10-7.05) (Table 3). MTHFR Gene The MTHFR polymorphism genotypes and allele frequencies are shown in Table 4. MTHFR CC genotype was the prominent polymorphism in all subjects, including controls. The pattern of genotypes was similar in all groups studied. There was no association between the MTHFR polymorphism and any site of vascular stenosis. ACE Gene The ACE polymorphism genotypes and allele frequencies are shown in Table 4. The ACE II genotype was the most common genotype in all 3 groups. ACE genotype
was not associated with the anatomical pattern of cerebral atherosclerosis.
Discussion In our study population, the APOE e4 allele was significantly associated with extracranial carotid artery atherosclerosis, but not with intracranial atherosclerosis (Table 2 and Fig 1). In addition, other factors, including the presence of diabetes, hypertension, and ischemic heart disease, were independently associated with extracranial carotid atherosclerosis (adjusted OR, 5.6, 5.1, and 3.0, respectively). These findings are in agreement with previous studies, mainly in Caucasians, that found an association between the APOE e4 allele and large-vessel atherosclerosis (especially extracranial carotid disease and increased intima-media thickness) in both asymptomatic patients and stroke patients.12-14,17-19 APOE also plays an important role in coronary artery atherosclerosis. Carriers of the e4 allele have an estimated 1.4-fold greater risk of developing coronary heart disease compared with e3 carriers.20,21 More importantly, although the APOE e4 allele was strongly associated with extracranial carotid and coronary heart disease, no association was found between this allele and isolated
Table 4. Comparison of MTHFR and ACE genotypes in controls, the EC group, and the IC group EC (n 5 58) MTHFR/ACE MTHFR CC TT CT Allele C Allele T ACE DD II ID Allele D Allele I
IC (n 5 83)
Controls (n 5 167), n (%)
n (%)
OR (95% CI)
n (%)
OR (95% CI)
121 (72.5) 9 (5.4) 37 (22.2) 279 (83.5) 55 (16.5)
44 (75.9) 5 (8.6) 9 (15.5) 97 (83.6) 19 (16.4)
1.19 (0.57-2.53) 1.66 (0.46-5.73) 0.65 (0.27-1.52) 1.01 (0.55-1.85) 0.99 (0.54-1.82)
59 (71.1) 4 (4.8) 20 (24.1) 138 (83.1) 28 (16.9)
0.93 (0.50-1.75) 0.89 (0.22-3.29) 1.12 (0.57-2.17) 0.97 (0.57-1.65) 1.03 (0.61-1.74)
35 (21) 68 (40.7) 64 (38.3) 134 (40.1) 200 (59.9)
6 (10.3) 28 (48.3) 24 (41.4) 36 (31.0) 80 (69.0)
0.44 (0.15-1.17) 1.36 (0.71-2.59) 1.14 (0.59-2.18) 0.67 (0.42-1.08) 1.49 (0.93-2.40)
22 (26.5) 32 (38.6) 29 (34.9) 73 (44.0) 93 (56.0)
1.36 (0.70-2.62) 0.91 (0.51-1.62) 0.86 (0.48-1.55) 1.17 (0.79-1.74) 0.85 (0.58-1.27)
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community-based study in Australia demonstrated an association between APOE genotype and low-density lipoprotein cholesterol (LDL-C) level.22 A meta-analysis of APOE genotypes with LDL-C level and coronary risk factors found that subjects with the e2/e2 genotype had a 31% lower mean LDL-C level than those with the e4/e4 genotype and a 20% lower coronary risk compared with the those with the more common e3/e3 genotype. A direct dose-response relationship among APOE genotype, LDL-C level, and coronary risk was noted, with subjects with the e4/e4 gentotype at highest risk, followed by those with the e3/e4 genotype.23,24 APOE genotype may partially explain the differing sites of predilection for atherosclerosis between Asians and Caucasians. The APOE genotypes and alleles in our study population were similar to those reported in previous studies from Japan and Taiwan.25,26 Although the e3/e3 genotype and e3 allele are common in both Asian and Caucasian populations, a significant difference in the proportion of e4 alleles has been noted.17,25-28 The e4 allele was found in only 5.7% of our control subjects and has been reported in 5%-10% in other Asian populations, compared with a prevalence of 15%-18% in Caucasians (Table 5).17,25-28 Because the APOE e4 allele is strongly associated with extracranial carotid disease, the lower frequency of the e4 allele in Asians may help explain the low prevalence of this disease in this population. Further investigation is still needed, however. Conflicting findings regarding the association between MTHFR polymorphisms and sites of atherosclerosis have been reported.29-33 In the present study, we found no significant difference in MTHFR polymorphisms and genotypes based on the presence or location of cerebral atherosclerosis (Table 4). The relationship between ACE gene polymorphisms and the location of vascular stenosis is controversial. Some studies have reported an association of the D genotype with increased carotid intima-media thickness 34,35 and significant carotid stenosis,36,37 but others have found no such association. Large studies in Japan and
Figure 1. Comparison of APOE genotypes between controls and the EC group (A) and between controls and the IC group (B).
intracranial atherosclerosis. Moreover, patients in the IC and EC groups had the same common atherosclerotic risk factors, such as diabetes and hypertension, with no significant difference in the risk factor profile between the two groups. Thus, we postulate that the development of intracranial atherosclerosis might be mediated or protected by other genetic factors or by the severity or duration of risk factors. The mechanism of atherosclerosis related to the APOE gene might be attributable to lipid and lipoprotein metabolism. APOE facilitates cholesterol homeostasis and is a component of most major lipoproteins. A large
Table 5. Comparison of APOE genotypes in control groups in Asian and Caucasian populations in previous studies Asian
Caucasian
APOE genotype
Thailand (n 5 167), %
Taiwan26 (n 5 1020), %
Rotterdam27 (n 5 5401), %
Australia28 (n 5 883), %
e2/e2 e2/e3 e2/e4 e3/e3 e3/e4 e4/e4 Allele e2 Allele e3 Allele e4
1.2 13.8 1.2 73.7 10.2 0 8.7 85.6 5.7
0.4 13.6 1.5 71.6 12.6 0.3 7.9 84.7 7.4
0.8 13.0 2.5 57.8 23.3 2.5 8.6 76.0 15.4
3 10 3 61 21 2 10 76 14
384
Australia found no correlation between carotid atherosclerosis and the I/D polymorphism.38 For intracranial atherosclerosis, a study of Chinese patients with type 2 diabetes found no association between ACE polymorphisms and stenosis of the middle cerebral artery.39 In our study, we found no association between ACE polymorphisms and either category of vascular stenosis studied (Table 4). We are aware of our study’s limitations. First, our study population is relatively small. However, the association between the APOE e4 allele and extracranial carotid stenosis was evident on both univariate and multivariate analyses, and our findings are consistent with those of previous studies. Second, we chose to study only the polymorphisms of 3 candidate genes. Because stroke is a complex disease, all of the polygenic interactions might not be readily apparent in a study of individual genes. Nonetheless, our study may provide clues to a help gain a deeper understanding of the genetic aspects of stroke.
Conclusion We found a significant association between the presence of the APOE e4 allele and extracranial carotid atherosclerosis in our Thai study population. We found no association between MTHFR or ACE polymorphisms with any site of cerebral atherosclerosis. The lower frequency of the APOE e4 allele in Asian populations may partially explain the low prevalence of extracranial carotid atherosclerosis in our study population. Acknowledgment: We gratefully acknowledge the support of the Neurological Society of Thailand.
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33. Kostulas K, Crisby M, Huang WX, et al. A methylenetetrahydrofolate reductase gene polymorphism in ischaemic stroke and in carotid artery stenosis. Eur J Clin Invest 1998;28:285-289. 34. Hosoi M, Nishizawa Y, Kogawa K, et al. Angiotensinconverting enzyme gene polymorphism is associated with carotid arterial wall thickness in non–insulindependent diabetic patients. Circulation 1996;94:704-707. 35. Kauma H, Paivansalo M, Savolainen MJ, et al. Association between angiotensin-converting enzyme gene polymorphism and carotid atherosclerosis. J Hypertens 1996;14:1183-1187. 36. Kostulas K, Huang WX, Crisby M, et al. An angiotensinconverting enzyme gene polymorphism suggests a genetic distinction between ischaemic stroke and carotid stenosis. Eur J Clin Invest 1999;29:478-483. 37. Pfohl M, Fetter M, Koch M, et al. Association between angiotensin I–converting enzyme genotypes, extracranial artery stenosis, and stroke. Atherosclerosis 1998;140: 161-166. 38. Mannami T, Katsuya T, Baba S, et al. Low potentiality of angiotensin-converting enzyme gene insertion/deletion polymorphism as a useful predictive marker for carotid atherogenesis in a large general population of a Japanese city: The Suita Study. Stroke 2001;32:1250-1256. 39. Thomas GN, Lin JW, Lam WW, et al. Middle cerebral artery stenosis in type II diabetic Chinese patients is associated with conventional risk factors but not with polymorphisms of the renin-angiotensin system genes. Cerebrovasc Dis 2003;16:217-223.
Ischemic stroke is a multifactorial disease with strong genetic elements. The purpose of this case-control study was to find relationships between apolipoprotein E (APOE), angiotensin-converting enzyme (ACE), and methylenetetrahydrofolate reductase (MTHFR) genotypes and atherosclerosis of the extracranial internal carotid and intracranial arteries in the Thai population. Patients aged .45 years with significant intracranial stenosis (IC group) or extracranial carotid artery stenosis (EC group) diagnosed by duplex ultrasound and/or computed tomography angiography (CTA) or magnetic resonance angiography (MRA) were studied. The control group comprised volunteers with no history of stroke and no evidence of significant cervicocerebral artery stenosis by ultrasound. Genomic DNA was extracted and genotyped for APOE isoforms, ACE insertion/deletion (I/D) polymorphism, and MTHFR C677T polymorphisms. There were 141 cases (83 in the IC group and 58 in the EC group) and 167 controls. The APOE e3/e4 genotype and APOE e4 allele were significantly associated with extracranial carotid artery stenosis (odds ratio, 2.55; 95% confidence interval, 1.07-6.05 and odds ratio, 2.85; 95% confidence interval, 1.35-5.99, respectively). These associations were not observed in patients with intracranial atherosclerosis. There was no significant association between ACE and MTHFR polymorphisms and stenosis at any site. In a multivariate model, sex, diabetes mellitus, hypertension, ischemic heart disease, and APOE e4 allele remained predictive of extracranial atherosclerosis. In our Thai population, the e4 allele in the APOE gene contributes to the genetic susceptibility of extracranial internal carotid atherosclerosis. The low prevalence of extracranial carotid stenosis in this population might result from low frequencies of the APOE e4 allele. Key Words: Apolipoprotein E—angiotensin-converting enzyme—methylenetetrahydrofolate reductase—intracranial stenosis—extracranial carotid stenosis. Ó 2012 by National Stroke Association
From the *Division of Neurology; †Division of Endocrinology, Department of Medicine, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand; and ‡Department of Neurology, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts. Received April 29, 2010; accepted October 10, 2010. Supported by the Neurological Society of Thailand. Address correspondence to Aurauma Chutinet, MD, Division of Neurology, Department of Medicine, Faculty of Medicine, Chulalongkorn University, Rama IV Road, Bangkok 10330, Thailand. E-mail: [email protected]. 1052-3057/$ - see front matter Ó 2012 by National Stroke Association doi:10.1016/j.jstrokecerebrovasdis.2010.10.002
Ischemic stroke is the leading cause of death and longterm disability worldwide.1 Among the different subtypes, atherosclerosis accounts for at least one-third of the cases.2,3 The common sites of cervicocerebral artery atherosclerosis are known to differ by race and ethnicity; for example, intracranial disease is more common in Asians and African Americans, whereas extracranial carotid stenosis is more common in Caucasians.4-8 Genetics, along with environmental factors, may play important roles in determining the site of atherogenesis and progression. A recent meta-analysis examining the association of genetic polymorphisms and susceptibility to
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stroke in Asian populations found significant associations of the 3 leading genes—the apolipoprotein E (APOE), angiotensin-converting enzyme (ACE), and methylenetetrahydrofolate reductase (MTHFR) genes—with stroke.9-11 Moreover, the APOE e4 allele has been found to be associated with intima-media thickness and stenosis of the extracranial carotid artery.12-14 None of those studies attempted to compare gene polymorphisms among various sites of vascular stenosis related to stroke, however. We postulated that genetic factors might play a role in determining the site of atherosclerosis, and accordingly, compared polymorphisms of APOE, ACE, and MTHFR in patients with significant extracranial atherosclerosis, those with intracranial atherosclerosis, and controls.
Patients and Methods Study Population This case-control study included Thai patients with significant ($50%) extracranial internal carotid and/or ($50%) intracranial atherosclerosis aged .45 years seen at King Chulalongkorn Memorial Hospital between March 2008 and February 2009. The degree of stenosis was determined by transcranial Doppler ultrasonography (TCD) and carotid duplex ultrasound (CDUS) and either computed tomography angiography (CTA) or magnetic resonance angiography (MRA). Patients with nonatherosclerotic causes of vascular stenosis (eg, dissection, vasculitis, moyamoya disease) were excluded. After informed consent was provided, baseline data (including demographic data, relevant past medical history, and family history of stroke and ischemic heart disease) were obtained, and a physical examination was performed. Blood samples for routine blood chemistry and genetic analyses were collected prospectively. The control group comprised healthy interval age-matched Thai volunteers who were members of an elders club at King Chulalongkorn Memorial Hospital. All control subjects were screened by questionnaire and physical examination to exclude previous stroke. TCD and CDUS were performed in all controls to exclude significant intracranial and extracranial carotid stenosis. This study was approved by the local Ethics Committee from the Institutional Review Board of the Faculty of Medicine, Chulalongkorn University (COA no. 676/2008; IRB no. 299/51). Written informed consent was obtained from each participant.
Evaluation for Arterial Stenosis To identify arterial stenosis, all enrolled patients were examined by TCD (Multidopp T, DWL-Compumedics, Germany) and CDUS (LOGIQ5 Expert, GE Healthcare, USA). In 90% of cases, MRA or CTA was performed and evaluated by a neuroradiologist to confirm the diagnosis of arterial stenosis. The interpreters of the MRA/CTA
were blinded to the CDUS and TCD results. The TCD criteria used to define $50% arterial stenosis were based on peak systolic flow velocity, as follows: .140 cm/s for middle cerebral artery, .120 cm/s for the anterior cerebral and internal carotid arteries, and .100 cm/s for the posterior cerebral, basilar, and vertebral arteries.15 Significant ($50%) extracranial stenosis on CDUS was defined according to previously published criteria.16 Patients with $50% stenosis of the extracranial internal carotid artery according to the ultrasound criteria were designated the EC group, and those with $50% intracranial artery stenosis without significant extracranial stenosis were designated the IC group.
Risk Factor Evaluation To identify the risk factors for atherosclerosis, a detailed history of established risk factors and medical history were obtained from each patient and prospectively recorded in the case record form. Baseline data included age, sex, presence of hypertension, diabetes mellitus, coronary heart disease, dyslipidemia, cigarette smoking, alcohol drinking, and history of stroke or coronary heart disease. Blood samples were collected for complete blood count, fasting plasma glucose, total cholesterol, triglyceride, high-density lipoprotein, and low-density lipoprotein.
Genotyping Genomic DNA was extracted from peripheral blood leukocytes by standard procedures with a DNA Mini Kit (Qiagen, Hilden, Germany). Genotyping for APOE isoforms, ACE I/D polymorphism, and MTHFR C677T polymorphism were analyzed. For APOE genotyping, DNA was amplified using two primers: 5’-ACAGAA TTCGCCCCGGCCTGGTACACAC-3’ and 5’-TAAGCTT GGCACGGCTGAAGGA-3’. Each amplification reaction contained 1 mg of leukocyte DNA, 1 pmol/mL of each primer, 10% dimethyl sulfoxide, and 2 units of Taq polymerase in a final volume of 30 mL. Each reaction mixture was heated at 95 C for 5 minutes, followed by 40 cycles of 95 C for 60 seconds, 65 C for 80 seconds, and 72 C for 80 seconds, with a final extension at 72 C for 7 minutes. The PCR products were treated with ExoSAP-IT (USP, Cleveland, OH) according to the protocols supplied by the manufacturer, and sent for direct sequencing to the Macrogen, Seoul, South Korea. For ACE I/D polymorphism, we used two primers, 5’-CTGGAGACCACTCCCATCATTTCT-3’ and 5’-GATGTG GCCATCACATTGGTCA GAT-3’, to detect a 190-bp fragment in the absence of insertion and a 490-bp fragment in the presence of insertion in the ACE gene. The amplicons were separated on 1% agarose gels and visualized using ethidium bromide staining and ultraviolet light. For MTHFR C677T, we used primers with the sequences 5’-ATCCCTATTGGC AGGTTAC-3’ and 5’-AGGACGGTGCGGTCAGAGTG-3’ in PCR reactions, and performed amplification using
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Table 1. Comparison of baseline characteristics and major risk factors in controls, the EC group, and the IC group Baseline characteristics and major risk factors Males, n (%) Age, years, mean 6 SD (95% CI) Diabetes, n (%) Hypertension, n (%) Ischemic heart disease, n (%) Dyslipidemia, n (%) Smoker, n (%) Alcohol drinking, n (%) Family history of ischemic heart disease, n (%)
Controls (n 5 167)
EC (n 5 58)
IC (n 5 83)
89 (53.3) 65.9 6 10.0 (64.4-67.4)
46 (79.3)* 72.4 6 8.9 (70.1-74.8)
53 (63.9) 65.2 6 10.5 (62.9-67.5)
13 (7.8) 92 (55.4) 10 (6.0) 151 (91.0) 10 (6.0) 24 (14.5) 12 (7.2)
27 (46.6)* 53 (91.4)* 15 (25.9)* 55 (94.8) 8 (14.5) 6 (10.9) 5 (9.1)
27 (33.3)* 71 (87.7)* 9 (11.1) 75 (92.6) 6 (7.5) 2 (2.5)* 6 (7.5)
*Statistically significant compared with controls.
initial denaturation at 94 C for 5 minutes, followed by 35 cycles of 94 C for 45 seconds, 60 C for 45 seconds, and 72 C for 45 seconds with a final extension at 72 C for 10 minutes. The C677T mutation creates a SchI recognition site, so digestion of the PCR product will generate two fragments (168 and 98 bp) that were size-fractionated on 1% agarose gels.
Statistical Analysis The risk factor profiles were compared between patients with significant stenosis of the extracranial carotid artery, those with significant stenosis of the intracranial arteries, and controls. Continuous variables were compared using an independent t test. Categorical variables were compared using the c2 test. Statistical significance was defined as a 5 0.05 by a two-tailed test. Logistic regression analysis was performed to identify the contributions of genetic polymorphisms and risk factors to the site of vascular stenosis.
Results Between March 2008 and February 2009, 308 subjects were included into the study (141 with significant stenosis
and 167 controls). Among the two groups of stenosis, 58 were in the EC group and 83 were in the IC group. Baseline characteristics are shown in Table 1. Males were predominant in all groups. The mean age was higher in the EC group. Diabetes mellitus, hypertension, ischemic heart disease, and smoking were significantly associated with stenosis. Male sex, diabetes mellitus, hypertension, and ischemic heart disease were significantly associated with extracranial carotid stenosis (P 5 .001, , .001, , .001, and , .001, respectively), whereas diabetes mellitus, hypertension, and alcohol consumption were significantly associated with intracranial artery stenosis (P , .001, , .001, and .003, respectively).
Genetic Study APOE Gene APOE genotypes and allele frequencies are shown in Table 2. The e3/e3 genotype was most prevalent in all groups and was present in 65%-75% of the cases. The e3/e4 genotype was more common in EC group (22.4%) compared with controls. The APOE e3 allele was the most common allele, found in .75% of subjects across
Table 2. Comparison of APOE genotypes in controls, the EC group, and the IC group EC (n 5 58)
IC (n 5 83)
APOE
Controls (n 5 167), n (%)
n (%)
OR (95% CI)
N (%)
OR (95% CI)
e2/e2 e3/e3 e4/e4 e2/e3 e2/e4 e3/e4 Allele e2 Allele e3 Allele e4
2 (1.2) 123 (73.7) 0 23 (13.8) 2 (1.2) 17 (10.2) 29 (8.7) 286 (85.6) 19 (5.7)
0 37 (67.3) 1 (1.7) 5 (8.6) 2 (3.4) 13 (22.4) 7 (6.0) 92 (79.3) 17 (14.6)
0 0.63 (0.32-1.25) 0 0.59 (0.16-1.75) 2.95 (0.29-30.10) 2.55 (1.07-6.05)* 0.68 (0.26-1.67) 0.64 (0.36-1.15) 2.85 (1.35-5.99)*
1 (1.2) 63 (75.9) 1 (1.2) 9 (10.8) 1 (1.2) 8 (9.6) 12 (7.2) 143 (86.1) 11 (6.6)
1.01 (0.20-5.02) 1.13 (0.59-2.17) 0 0.76 (0.31-1.84) 1.01 (0.20-5.02) 0.94 (0.35-2.44) 0.82 (0.38-1.73) 1.04 (0.59-1.85) 1.18 (0.51-2.68)
*Statistically significant compared with controls.
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Table 3. Multiple logistic regression analysis between controls and the EC group Risk factor
Crude OR (95% CI)
Adjusted OR (95% CI)
Female sex Age Diabetes mellitus Hypertension Ischemic heart disease Smoker APOE e3/e4 APOE e4 allele
0.3 (0.15-0.60)* 4.7 (1.76-12.35)* 10.2 (4.76-22.05)* 8.5 (3.24-22.42)* 5.4 (2.28-12.97)* 2.6 (0.99-7.11) 2.5 (1.07-6.05)* 2.8 (1.35-5.99)*
0.3 (0.15-0.82)* 3.2 (0.95-10.58) 5.6 (2.32-13.44)* 5.1 (1.76-14.81)* 3.0 (1.04-8.87)* – – 2.8 (1.10-7.05)*
*Statistically significant.
all groups. APOE e3/e4 genotype and the e4 allele were significantly associated with extracranial carotid stenosis compared with controls (odds ratio [OR], 2.55; 95% confidence interval [CI], 1.07-6.05 vs OR, 2.85; 95% CI, 1.35-5.99) (Table 2). Logistic regression analysis was performed to determine the predictors of extracranial carotid stenosis. On multivariate regression, sex, diabetes mellitus, hypertension, ischemic heart disease, and APOE e4 allele were independently associated with extracranial carotid stenosis (APOE e4 allele: OR, 2.8; 95% CI, 1.10-7.05) (Table 3). MTHFR Gene The MTHFR polymorphism genotypes and allele frequencies are shown in Table 4. MTHFR CC genotype was the prominent polymorphism in all subjects, including controls. The pattern of genotypes was similar in all groups studied. There was no association between the MTHFR polymorphism and any site of vascular stenosis. ACE Gene The ACE polymorphism genotypes and allele frequencies are shown in Table 4. The ACE II genotype was the most common genotype in all 3 groups. ACE genotype
was not associated with the anatomical pattern of cerebral atherosclerosis.
Discussion In our study population, the APOE e4 allele was significantly associated with extracranial carotid artery atherosclerosis, but not with intracranial atherosclerosis (Table 2 and Fig 1). In addition, other factors, including the presence of diabetes, hypertension, and ischemic heart disease, were independently associated with extracranial carotid atherosclerosis (adjusted OR, 5.6, 5.1, and 3.0, respectively). These findings are in agreement with previous studies, mainly in Caucasians, that found an association between the APOE e4 allele and large-vessel atherosclerosis (especially extracranial carotid disease and increased intima-media thickness) in both asymptomatic patients and stroke patients.12-14,17-19 APOE also plays an important role in coronary artery atherosclerosis. Carriers of the e4 allele have an estimated 1.4-fold greater risk of developing coronary heart disease compared with e3 carriers.20,21 More importantly, although the APOE e4 allele was strongly associated with extracranial carotid and coronary heart disease, no association was found between this allele and isolated
Table 4. Comparison of MTHFR and ACE genotypes in controls, the EC group, and the IC group EC (n 5 58) MTHFR/ACE MTHFR CC TT CT Allele C Allele T ACE DD II ID Allele D Allele I
IC (n 5 83)
Controls (n 5 167), n (%)
n (%)
OR (95% CI)
n (%)
OR (95% CI)
121 (72.5) 9 (5.4) 37 (22.2) 279 (83.5) 55 (16.5)
44 (75.9) 5 (8.6) 9 (15.5) 97 (83.6) 19 (16.4)
1.19 (0.57-2.53) 1.66 (0.46-5.73) 0.65 (0.27-1.52) 1.01 (0.55-1.85) 0.99 (0.54-1.82)
59 (71.1) 4 (4.8) 20 (24.1) 138 (83.1) 28 (16.9)
0.93 (0.50-1.75) 0.89 (0.22-3.29) 1.12 (0.57-2.17) 0.97 (0.57-1.65) 1.03 (0.61-1.74)
35 (21) 68 (40.7) 64 (38.3) 134 (40.1) 200 (59.9)
6 (10.3) 28 (48.3) 24 (41.4) 36 (31.0) 80 (69.0)
0.44 (0.15-1.17) 1.36 (0.71-2.59) 1.14 (0.59-2.18) 0.67 (0.42-1.08) 1.49 (0.93-2.40)
22 (26.5) 32 (38.6) 29 (34.9) 73 (44.0) 93 (56.0)
1.36 (0.70-2.62) 0.91 (0.51-1.62) 0.86 (0.48-1.55) 1.17 (0.79-1.74) 0.85 (0.58-1.27)
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community-based study in Australia demonstrated an association between APOE genotype and low-density lipoprotein cholesterol (LDL-C) level.22 A meta-analysis of APOE genotypes with LDL-C level and coronary risk factors found that subjects with the e2/e2 genotype had a 31% lower mean LDL-C level than those with the e4/e4 genotype and a 20% lower coronary risk compared with the those with the more common e3/e3 genotype. A direct dose-response relationship among APOE genotype, LDL-C level, and coronary risk was noted, with subjects with the e4/e4 gentotype at highest risk, followed by those with the e3/e4 genotype.23,24 APOE genotype may partially explain the differing sites of predilection for atherosclerosis between Asians and Caucasians. The APOE genotypes and alleles in our study population were similar to those reported in previous studies from Japan and Taiwan.25,26 Although the e3/e3 genotype and e3 allele are common in both Asian and Caucasian populations, a significant difference in the proportion of e4 alleles has been noted.17,25-28 The e4 allele was found in only 5.7% of our control subjects and has been reported in 5%-10% in other Asian populations, compared with a prevalence of 15%-18% in Caucasians (Table 5).17,25-28 Because the APOE e4 allele is strongly associated with extracranial carotid disease, the lower frequency of the e4 allele in Asians may help explain the low prevalence of this disease in this population. Further investigation is still needed, however. Conflicting findings regarding the association between MTHFR polymorphisms and sites of atherosclerosis have been reported.29-33 In the present study, we found no significant difference in MTHFR polymorphisms and genotypes based on the presence or location of cerebral atherosclerosis (Table 4). The relationship between ACE gene polymorphisms and the location of vascular stenosis is controversial. Some studies have reported an association of the D genotype with increased carotid intima-media thickness 34,35 and significant carotid stenosis,36,37 but others have found no such association. Large studies in Japan and
Figure 1. Comparison of APOE genotypes between controls and the EC group (A) and between controls and the IC group (B).
intracranial atherosclerosis. Moreover, patients in the IC and EC groups had the same common atherosclerotic risk factors, such as diabetes and hypertension, with no significant difference in the risk factor profile between the two groups. Thus, we postulate that the development of intracranial atherosclerosis might be mediated or protected by other genetic factors or by the severity or duration of risk factors. The mechanism of atherosclerosis related to the APOE gene might be attributable to lipid and lipoprotein metabolism. APOE facilitates cholesterol homeostasis and is a component of most major lipoproteins. A large
Table 5. Comparison of APOE genotypes in control groups in Asian and Caucasian populations in previous studies Asian
Caucasian
APOE genotype
Thailand (n 5 167), %
Taiwan26 (n 5 1020), %
Rotterdam27 (n 5 5401), %
Australia28 (n 5 883), %
e2/e2 e2/e3 e2/e4 e3/e3 e3/e4 e4/e4 Allele e2 Allele e3 Allele e4
1.2 13.8 1.2 73.7 10.2 0 8.7 85.6 5.7
0.4 13.6 1.5 71.6 12.6 0.3 7.9 84.7 7.4
0.8 13.0 2.5 57.8 23.3 2.5 8.6 76.0 15.4
3 10 3 61 21 2 10 76 14
384
Australia found no correlation between carotid atherosclerosis and the I/D polymorphism.38 For intracranial atherosclerosis, a study of Chinese patients with type 2 diabetes found no association between ACE polymorphisms and stenosis of the middle cerebral artery.39 In our study, we found no association between ACE polymorphisms and either category of vascular stenosis studied (Table 4). We are aware of our study’s limitations. First, our study population is relatively small. However, the association between the APOE e4 allele and extracranial carotid stenosis was evident on both univariate and multivariate analyses, and our findings are consistent with those of previous studies. Second, we chose to study only the polymorphisms of 3 candidate genes. Because stroke is a complex disease, all of the polygenic interactions might not be readily apparent in a study of individual genes. Nonetheless, our study may provide clues to a help gain a deeper understanding of the genetic aspects of stroke.
Conclusion We found a significant association between the presence of the APOE e4 allele and extracranial carotid atherosclerosis in our Thai study population. We found no association between MTHFR or ACE polymorphisms with any site of cerebral atherosclerosis. The lower frequency of the APOE e4 allele in Asian populations may partially explain the low prevalence of extracranial carotid atherosclerosis in our study population. Acknowledgment: We gratefully acknowledge the support of the Neurological Society of Thailand.
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