Arachidonate 5-lipoxygenase-activating protein (ALOX5AP) gene rs4073259 polymorphism not associated with ischemic stroke in the Northeastern Chinese Han population☆

Clinical Neurology and Neurosurgery 119 (2014) 64–69

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Arachidonate 5-lipoxygenase-activating protein (ALOX5AP) gene rs4073259 polymorphism not associated with ischemic stroke in the Northeastern Chinese Han population夽夽 RuYou Zhang a,1 , XiJuan Guo b,1 , XiaoYing Li c , Wei Liu c , YanQing Peng c , XueSong Han c , JiaWei Tian c , LiTao Sun c,∗ , Yan Liu d,∗∗ a

Department of Neurosurgery, The 2nd Affiliated Hospital of Harbin Medical University, Harbin, China Department of Ultrasound, The Fourth Hospital of Shijiazhuang, ShiJiaZhuang, China c Department of Ultrasound, The 2nd Affiliated Hospital of Harbin Medical University, Harbin, China d Department of Biostatistics, School of Public Health, Harbin Medical University, Harbin, China b

a r t i c l e

i n f o

Article history: Received 24 May 2013 Received in revised form 17 December 2013 Accepted 4 January 2014 Available online 15 January 2014 Keywords: SNP Ischemic stroke Case and control study

a b s t r a c t Objective: Although recent evidence has implicated that 5-lipoxygenase activating protein (ALOX5AP) gene is associated with ischemic stroke (IS) risk, the underlying molecular mechanism remains to be defined. This study aimed to investigate the role of ALOX5AP rs4073259 in ischemic stroke in a Northeastern Chinese Han population. Methods: A total of 501 IS patients and 497 healthy controls were enrolled for polymerase chain reaction (PCR) and ligase detection reaction (LDR) analysis of ALOX5AP rs4073259 single nucleotide polymorphism (SNP). Results: There were no statistically significant differences in ALOX5AP rs4073259 allele and genotype frequencies between IS or subtypes of IS and controls. There was no significant difference in genotype and allele frequencies of atherosclerosis degree between ischemic subjects with carotid artery plaque or absence. However, total cholesterol (TC), low-density lipoprotein (LDL), and high-density lipoprotein (HDL) in women patients were significantly higher than those in men (p = 0.015, 0.000, and 0.008, respectively). Total homocysteine (tHcy) was higher in men patients than that in women (p = 0.021). Conclusion: There was no statistically significant association of ALOX5AP rs4073259 SNP with ischemic stroke in this northeastern Chinese Han population living in Heilongjiang province, China. © 2014 The Authors. Published by Elsevier B.V. All rights reserved.

1. Introduction Ischemic stroke (IS) is one of the leading causes of death and adult disability in the world [1,2]. Accumulating evidence has indicated that ischemic stroke is a complicated clinical syndrome with diverse etiologies, including genetic predisposition

夽 This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-No Derivative Works License, which permits non-commercial use, distribution, and reproduction in any medium, provided the original author and source are credited. ∗ Corresponding author at: Department of Ultrasound, The 2nd Affiliated Hospital of Harbin Medical University, 246, Xuefu Road, Harbin 150081, China. Tel.: +86 0451 86605811; fax: +86 0451 86675845. ∗∗ Corresponding author at: Department of Biostatistics, School of Public Health, Harbin Medical University, 194, Xuefu Road, Harbin 150081, China. Tel.: +86 0451 87502937; fax: +86 0451 87502831. E-mail addresses: [email protected] (L. Sun), [email protected] (Y. Liu). 1 These authors contributed equally to this work.

and environmental factors [3–5]. Recent studies have also shown that inflammation plays an important role in pathogenesis of atherosclerosis, which is the major pathological basis of ischemic stroke and cardiovascular disease [6–11]. Leukotrienes (LTs) are the key components in inflammatory reactions and secreted by various types of inflammatory cells, which are also implicated in initiation and progression of atherosclerosis [6,12,13]. 5-lipoxygenase activating protein (FLAP) and 5-lipoxygenase pathway (5-LO) are the key factors involved in the leukotrienes production [14,15]. Following Ca+ -dependent cell activation, arachidonic acid is released from the cell membrane phospholipids by cytosolic phospholipase A2 for synthesis of 5-LO by FLAP [16]. During this process, arachidonate is converted into a unstable intermediate LTA4 [17] and conjunction of LTA4 with glutathione by LTC4 synthase forms LTC4. LTC4 is then cleaved by gamma-glutamyl transpeptidase to LTD4, which is then metabolized by a dipeptidase to LTE4 [18,19]. The main pathophysiological effect of these cysteinyl-LTs includes leucocytes activation, monocyte diapedesis across the endothelial barrier, and increasing permeability of blood vessels [20].

0303-8467/$ – see front matter © 2014 The Authors. Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.clineuro.2014.01.004

R. Zhang et al. / Clinical Neurology and Neurosurgery 119 (2014) 64–69

The role of FLAP and 5-LO in production of leukotrienes could be involved in pathogenesis of atherosclerosis. So it is reasonable and interesting to investigate the association of FLAP and 5-LO with IS. DeCODE genetic group first showed a single ALOX5AP haplotype consisted of four single nucleotide acids, termed Hap A, could increase risk of myocardial infarction and ischemic stroke by nearly two folds in the Iceland population [21]. The same research group also found another haplotype, i.e., Hap B, was closely associated with the incidence of myocardial infarction in individuals from United Kingdom [22]. In contrast, another study found that ALOX5AP polymorphism had no association with MRI-defined brain infarcts [23]. However, there were only a few studies reporting the association of ALOX5AP rs4073259 with IS [24,25]. Thus, in this study we mainly focused on investigation of ALOX5AP rs4073259 association with IS risk in a northeastern Chinese Heilongjiang province Han population.

2. Subjects and methods 2.1. Subjects A total of 501 patients with unrelated IS of the northeastern Chinese Han population (331 males, 170 females) were included in this study. These patients have lived in Heilongjiang province for more than 20 years and were enrolled in this study from the Department of Neurology, The Second Hospital affiliated to Harbin Medical University (Harbin, Heilongjiang, China) between April 2011 and May 2012. All patients underwent the computed tomography (CT), magnetic resonance imaging (MRI), or both in the head within one week after hospital admission. Among these 501 patients, 210 patients underwent magnetic resonance angiography (MRA) or computed tomography angiography (CTA) to evaluate severity of cerebral atherosclerosis. Eighty-seven patients received carotid duplex sonography and showed there were two groups of patients, i.e., Group A was without carotid artery atherosclerosis plaque and Group B was with carotid atherosclerosis plaque (e.g., hard plaque, combined plaques, and soft plaques). The patients undergone MRA or CTA were divided into two groups, i.e., Group C was with intracranial artery stenosis rate of ≤50% and Group D was with intracranial artery stenosis rate of >50%. Transthoracal echocardiography was obtained in 150 patients. According to the criteria in the Trial of Org 10,172 in Acute Stroke Treatment (TOAST) classification [26], these patients were further classified into five subgroups, i.e., large artery atherosclerosis, n = 81 (16.2%); lacunar stroke, n = 310 (61.9%); large artery atherosclerosis and lacunar stroke (combination type), n = 104 (20.8%); cardiogenic stroke, n = 2 (0.4%); the etiology of stroke remained unclear despite of different diagnostic efforts, n = 4 (0.8%). The subtype analysis was limited to large artery atherosclerosis, lacunar stroke, and combination type because of small sample sizes of other subtypes. Furthermore, we also recruited 497 healthy unrelated individuals as controls (300 males, 197 females) from Health Medical Center, The Second Hospital affiliated to the Harbin Medical University. All control subjects had no history of stroke, other central nervous system disorders, cardiovascular disease, cancer, endocrine diseases, infectious diseases, trauma, or surgery history. After neurological examination, there were no focal signs detected in these control subjects. Among them, approximate 80 subjects received brain CT scan and there were no positive disorders detected. A questionnaire was designed to collect detailed information about risk factors of stroke, including gender, age, hypertension, diabetes mellitus, hyperlipidemia, coronary heart disease, stroke history, arterial fibrillation, smoking, and alcohol abusing for both cases and controls. This study was approved by the local ethics

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committee and each subject enrolled in this study was informed and consented. 2.2. Genotyping Peripheral venous blood was withdrawn form each studied subject for genotyping of ALOX5AP rs4073259 polymorphism, which was performed by Shanghai BioWing Applied Biotechnology Company (http://www.biowing.com.cn) using a ligase detection reaction (LDR) [27]. The primers used to the target DNA sequence were 5 -CCAAAGGCTTCACCTCTGAT-3 and 5 CGGCACATGAAAACAGCAC-3 . In brief, genomic DNA was extracted from EDTA blood using a standard protocol [28] and these DNA samples were amplified by PCR in a 20 ␮l reaction mixture, which contained 1× PCR buffer, 0.6 ␮l Mg++ (3.0 mmol/L), 50 ng of genomic DNA, 0.4 ␮l each primer, 2 ␮l deoxynucleotide triphosphates (2.0 mmol/L), 0.3 ␮l of 1U Qiagen HotStarTaq Polymerase (Qiagen, Germany) and 4 ␮l of 1× Q-solution. The LDR was performed in a 10 ␮l reaction mixture containing 1× buffer, 12.5 pmol/␮l of each probe mix, 0.05 ␮l of 2U Taq DNA ligase (New England Biolabs, Ipswich, MA, USA) and ≥1 ␮l of 100 ng/␮l multiPCR product. Quality of the genotyping was evaluated by testing blind blood duplicates (49). 2.3. Statistics analysis Allele and genotype frequencies were calculated for each locus and the Hardy–Weinberg equilibrium was performed using the 2 test with SPSS 20.0 for windows software (SPSS, Chicago, IL, USA). The association between phenotype and genotype was analyzed by the 2 test. Logistic regression was used to adjust the bias of classic stroke risk factors, such as age, BMI, blood pressure, plasma glucose level, lipid profile, cigarette smoking, and alcohol intake. A p-value of less than 0.05 was considered to be statistically significant. 3. Results 3.1. Characteristics of subjects The clinical characteristics for the 501 IS patients and 497 healthy control subjects used in this study are shown in Table 1. Particularly, there were significant differences observed between the IS and control group for age, BMI, SBP, DBP, hypertension, DM, lipids, smoking, and alcohol consumption. 3.2. Genotype and allele frequencies The characteristics of allele frequency and genotype frequency were determined for rs4073259 in ALOX5AP promoter region and the results was summarized in Table 2. In brief, the A allele was shown at a frequency of 53.6% in cases vs. 54.7% in controls. The G allele was found at a frequency of 46.4% in cases vs. 45.3% in controls. There was no statistically significant difference in genotype and allele frequencies between patients and controls. Using logistics regression analysis to adjust the bias of conventional stroke risk factors, there was also no association between the case and control (p = 0.834 and 0.999, respectively). The allele and genotype frequencies of ALOX5AP rs4073259 did not significantly differ between the case and control using Hardy–Weinberg equilibrium analysis (p = 0.237). 3.3. Subtype analysis of IS The subtype analysis of IS was performed using the groups of large artery atherosclerosis, lacunar stroke and combination type. A summary of the characteristics of allele frequency and genotype

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Table 1 Clinical characteristics of ischemic stroke patients and healthy controls (mean ± standard deviation).

Age, years Men, n (%) BMI, kg/m2 SBP, mm Hg DBP, mm Hg Glucose, mmol/L TC, mmol/L TG, mmol/L HDL, mmol/L LDL, mmol/L ApoA, g/L ApoB, g/L Lip, ␮ml/L tHcy, ␮ml/L Cigarette smoker, n (%) Alcohol intake, n (%) Hypertension, n (%) Diabetes, n (%) Cardiopathy, n (%)

Controls (n = 497)

Cases (n = 501)

p-Value

53.12 ± 8.60 300 (60.4) 23.39 ± 3.28 115.64 ± 11.49 78.85 ± 6.46 5.13 ± 2.04 4.41 ± 0.51 0.99 ± 0.31 1.47 ± 0.27 2.67 ± 0.45

58.69 ± 10.17 331 (66.07) 24.811 ± 3.33 148.22 ± 23.46 89.62 ± 13.45 6.8026 ± 3.10 5.3082 ± 3.39 1.8884 ± 1.47 1.2783 ± 0.46 2.8770 ± 0.82 1.10 ± 0.214 0.81 ± 0.24 2.14 ± 0.45 18.64 ± 9.17 245 (48.90) 191 (38.12) 334 (66.67) 113 (22.55) 68 (13.57)

0.000 0.062 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000

121 (24.35) 134 (26.96) 0 (0) 0 (0) 0 (0)

Table 4 Association of ALOX5AP rs4073259 between patients with or without carotid artery atherosclerosis plaque.

AA AG GG A allele frequency G allele frequency

Group A

Group B

p-Value

5 (5.7) 6 (6.9) 4 (4.6) 9.2 42.0

22 (25.3) 29 (33.3) 21 (24.1) 8.0 40.8

0.971 0.793

Group A was without carotid artery atherosclerosis plaque, whereas Group B had carotid atherosclerosis plaque (including hard plaque, combined plaques, and soft plaques).

Table 5 Association of ALOX5AP rs4073259 between Group C and D patients. 0.000 0.000

Notes: BMI, body mass index; SBP, systolic blood pressure; DBP, diastolic blood pressure; TC, total cholesterol; TG, triglycerides; HDL, high-density lipoprotein cholesterol; LDL, low density lipoprotein; ApoA, apolipoprotein A; ApoB, apolipoprotein B; Lip, lipoprotein a; tHcy, total homocystein.

AA AG GG A allele frequency G allele frequency

Group C

Group D

p-Value

35 (16.7) 58 (27.6) 28 (13.3) 30.5 27.1

27 (12.9) 40 (19.0) 22 (10.5) 22.4 20.0

0.911 0.986

Group C patients had ≤50% of an intracranial artery stenosis rate, whereas Group D patients had more than 50% of an intracranial artery stenosis rate.

3.5. Association of stroke patients with different severity of atherosclerosis frequency in subtypes of IS are shown in Table 3. In particular, the A allele was found at a frequency of 53.7% in large artery atherosclerosis, 54.0% in lacunar stroke, and 52.4% in combination type. The G allele was found at a frequency of 46.3% in large artery atherosclerosis, 46.0% in lacunar stroke, and 48.6% in combination type. There was no statistically significant difference in genotype and allele frequencies among these IS subtypes.

MRA or CTA were utilized as a tool to evaluate severity of cerebral atherosclerosis. Based on the established grading system, we divided 210 IS patients who underwent MRA or CTA into two subgroups, i.e., Group C patients had ≤50% of an intracranial artery stenosis rate, whereas Group D patients had >50% of an intracranial artery stenosis rate. The frequencies of ALOX5AP G/A genotype or allele of rs4073259 had not difference between these two groups of patients (p = 0.911 and 0.986, respectively; Table 5).

3.4. Association of stroke patients with carotid artery atherosclerosis plaque

3.6. Association of genotype and lipid profile of patients

We also evaluated the association of ALOX5AP rs4073259 SNP with existence of carotid artery atherosclerosis plaque in ischemic subjects (Table 4). No significant associations were observed for the genotype and allele frequencies between ischemic subjects with carotid artery atherosclerosis plaque (p = 0.971 and 0.793, respectively).

We then associated ALOX5AP genotypes with lipid profiles of these 501 ischemic stroke patients and analyzed the lipid profiles between male and female patients. The lipid profiles of the patients were classified as low, normal, and high according to the previously defined normal range (TC, 3.8–5.17 mmol/L; TG, 0.56–1.70 mmol/L; LDL, 0.45–3.15 mmol/L; HDL, 1.04–1.70 mmol/L; ApoA, 1.0–1.6 g/L;

Table 2 ALOX5AP rs4073259 allele and genotype frequencies between case and control. rs4073259

Allele (%) A

Case Control a

53.6 54.7

p1

p1a

Genotype, n (%)

G

AG

AA

46.4 45.3

0.611

147 155 (31.2)

0.999

243 (48.5) 234 (47.1)

p2

p2a

H-W p3

0.816

0.834

0.237

GG 111 (22.2) 108 (21.7)

Adjusted by age, sex, BMI, SBP, DBP, Glu, TC, TG, LDL, HDL, hypertension, diabetes, tobacco smoking, and alcohol consumption.

Table 3 ALOX5AP rs4073259 allele and genotype frequencies in IS subtypes. rs4073259

Allele (%)

Control (n = 497) Atherothrombosis (n = 81) Lacunar (n = 310) Combination (n = 104)

54.7 53.7 54.0 52.4

p1

A 0.799 0.829 0.456

Genotype, n (%)

p2

AA

AG

155(31.2) 22 (27.2) 94 (30.3) 28 (26.92)

234(47.1) 43 (53.1) 147 (47.4) 51 (49.04)

GG 108(21.7) 16 (19.8) 69 (22.3) 25 (24.04)

0.601 0.963 0.674

R. Zhang et al. / Clinical Neurology and Neurosurgery 119 (2014) 64–69

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Table 6 Association of ALOX5AP rs4073259 polymorphism with lipid profile in IS patients. rs4073259 A/G

TC Low Normal High TG Low Normal High HDL Low Normal High LDL Normal High ApoA Low Normal High Apo B Low Normal High Lip Low Normal High tHcy Normal High

Sex

AA

AG

GG

p1

Male

Female

p2

19 (4.0) 56 (11.8) 68 (14.3)

21 (4.4) 99 (20.8) 107 (22.5)

11 (2.3) 48 (10.1) 47 (9.9)

0.711

36 (7.6) 148 (31.1) 133 (27.9)

15 (3.2) 55 (11.6) 89 (18.7)

0.015

3 (0.6) 77 (16.2) 63 (13.2)

4 (0.8) 128 (26.9) 95 (20)

1 (0.2) 61 (12.8) 44 (9.2)

0.939

5 (1.1) 173 (36.3) 139 (29.2)

3 (0.6) 93 (19.5) 63 (13.2)

0.671

36 (7.6) 100 (21.0) 7 (1.5)

56 (11.8) 147 (30.9) 24 (5.0)

27 (5.7) 71 (14.9) 8 (1.7)

0.421

93 (19.5) 199 (41.8) 25 (5.3)

26 (5.4) 119 (25.0) 14 (2.9)

0.008

88 (18.5) 55 (11.6)

155 (32.6) 72 (15.1)

80 (16.8) 26 (5.5)

0.065

232 (48.7) 85 (17.9)

91 (19.1) 68 (14.3)

0.000

49 (10.9) 83 (18.4) 0 (0)

83 (18.4) 130 (28.8) 2 (0.4)

34 (7.5) 67 (14.9) 3 (0.7)

0.253

118 (26.2) 179 (39.7) 2 (0.4)

48 (10.6) 101 (22.4) 3 (0.7)

0.139

20 (4.4) 95 (21.1) 17 (3.8)

36 (8.0) 161 (35.7) 18 (4.0)

15 (3.3) 77 (17.1) 12 (2.7)

0.712

46 (10.2) 228 (50.6) 25 (5.5)

25 (5.5) 105 (23.3) 22 (4.9)

0.111

41 (9.1) 89 (19.8) 2 (0.4)

57 (12.7) 151 (33.6) 6 (1.3)

29 (6.4) 72 (16.0) 3 (0.7)

0.853

90 (20.0) 203 (45.1) 5 (1.1)

37 (8.2) 109 (24.2) 6 (1.3)

0.171

38 (12.5) 46 (15.1)

72 (23.7) 75 (24.7)

36 (11.8) 37 (12.2)

0.834

88 (28.9) 115 (37.8)

58 (19.1) 43 (14.1)

0.021

Values are expressed as total number and percentage in the parentheses.

ApoB, 0.6–1.1 g/L; Lip, 1.9–3.2 ␮ml/L; and total homocysteine, 5.08–15.39 ␮ml/L). ALOX5AP rs4073259 polymorphism was not associated with total cholesterol (p = 0.711), Triglycerides (p = 0.939), HDL (p = 0.421), LDL cholesterol (p = 0.065), Apolipoprotein A (p = 0.253), ApoB (p = 0.712), Lip (p = 0.853), and tHcy (p = 0.834). TG, ApoA, ApoB, and Lip were not different between male and female patients (p = 0.671, 0.139, 0.111, and 0.171, respectively). However, TC, LDL, HDL and tHcy were different between male and female patients, i.e., TC, LDL and HDL were significantly higher in female patients than in male patients (p = 0.015, 0.000, and 0.008, relatively), while tHcy was higher in male patients than that in female patients (p = 0.021; Table 6). 4. Discussion In this study, we investigated the association between ALOX5AP rs4073259 polymorphism and IS susceptibility. However, we did not find a statistical difference in gender distribution between the case and the control, although the proportion of male patients in the case group (60.4%) is higher than that of women patients (39.6%). The median age, body mass index (BMI), blood lipids, glucose, and blood pressure levels, tobacco, and alcohol consumption were higher than those in the controls, which is consistent with the epidemiological findings. A previous study demonstrated that many factors were contributed to IS and intracerebral haemorrhagic stroke; for example, history of hypertension, current smoker, waist-to-hip ratio, diet risk score, physical activity, diabetes mellitus, alcohol intake, psychosocial stress and depression, cardiac alteration, and ratio of apolipoproteins B to A1 [4]. Furthermore, there was no statistical association of ALOX5AP allele and genotype frequencies between case or IS subtype and controls after a multivariate logistic regression analysis. After adjusting the bias of classical stroke risk factors, the results revealed that G/A genotype

of ALOX5AP rs4073259 did not contribute to IS risk. There was no statistically significant difference in ALOX5AP genotype and allele frequencies between the IS subtype and controls. However, two previous studies of different ethnic patients showed that ALOX5AP rs4073259 was associated with risk of IS [24,25], i.e., Lohmussaar et al. [24] showed that there was a stronger association of ALOX5AP rs4073259 between IS in male patients than that in female patients in Iceland population (odds ratio, 1.26; 95% CI 1.03–1.54; p = 0.024). Zhang et al. [25] analyzed ALOX5AP polymorphism in 236 patients with a history of cerebral infarction and 219 healthy subjects without a history of cerebral infarction or cardiovascular disease and family history of cerebrovascular diseases for three generations, which included individuals in the Han population whose families have lived in the Northeastern part of China, including Heilongjiang, Jilin, and Liaoning, for more than three generations. They found that ALOX5AP rs4073259 A allele was associated with an increased risk of ischemic stroke, whereas ALOX5AP rs4073259 GG was associated with a decreased risk. In addition, Ji et al. [29] showed that ALOX5AP promoter region-581 582 Ins A, an insertion and deletion polymorphism, was a novel genetic variant for ischemic stroke risk. Furthermore, a haplotype-based analysis for ALOX5AP haplotype AA of block 2 involving rs10507391 and rs12429692, and rs9579646 AG genotype was associated with a decreased risk of stroke in Eastern Chinese Han population [30]. However, in this study, we did not find any association of ALOX5AP rs4073259 between the case and control. We also did not observe the association of A allele with the increased risk of ischemic stroke in the Han population, which lived in Heilongjiang province more than 20 years. Our data failed to replicate all of these previous findings. The underlying reason is unknown, but might be due to different populations, sampling strategies, or phenotype heterogeneity. Another possibility is that etiologies of stroke in this study and previous studies might have been different. For example, in this study, IS patients with lacunar infarction had highest proportion

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(61.9%), while IS incidence with unknown etiologies was lowest (0.8%). The latter was much lower than that reported in the literature [31], but it was consistent with another Chinese study reported by Wu et al. [32]. However, the discrepancy of the rates between Chinese and other ethnic population remains unknown, which may lead to a future collaborative study on this. Furthermore, although 80 subjects of our healthy controls without cerebrovascular diseases who underwent brain CT scan did not show any positive findings, we could not ensure that brain CT scan without positive finding in all of these healthy controls, thus, silent strokes cannot be ruled out. We speculate that environmental factors may contribute to expression and activity of ALOX5AP. Heilongjiang is localized at a high latitude area where residents grow accustom to high salt and fat diets, which are risk factors for hypertension and promote a lacunar stroke. In addition, detection of a single gene polymorphism could only associate with a modest effect, but multiple SNPs in multiple genes plus a haplotype analysis may have more powerful to associate with IS risk. Furthermore, this study further explored the association of carotid plaques and degree of cerebral atherosclerosis with ALOX5AP rs4073259 polymorphism. We found that the G/A genotype of rs4073259 was not a risk factor in developing atherosclerosis in ischemic subjects. Jin et al. [33] showed that frequency of SG13S114 AA genotype and A allele was significantly higher in atherothrombotic cerebral infarction patients with vulnerable plaques than those with stable plaques; thus, and they deduced that A allele may be a risk factor of vulnerable plaques. However, in this study, we did not find that ALOX5AP rs4073259 associated with carotid artery atherosclerosis plaque. Based on the small number of patients who underwent to carotid ultrasound examination and cerebral angiography, we cannot rule out contribution of ALOX5AP rs4073259 to atherosclerosis in ischemic stroke patients. It has been reported that various gene polymorphisms affect lipids and tHcy profile, although a previous study by Shin et al. [34] showed that there was no association between PON gene polymorphisms and stroke, but PON1-55 L allele elevated the plasma concentration of total homocysteine, PON2-148A and 311 S alleles were associated with higher plasma total and LDL cholesterol, and ApoB, PON2-148G elevated plasma concentration of total and HDL cholesterol and ApoA1. Another study showed that that the HindIII polymorphism of LPL was associated with ischemic stroke and elevated levels of plasma triglycerides and reduced HDL levels. This gene polymorphism was associated with intracranial large artery atherosclerosis that is the most frequent subtype in Andhra Pradesh [35]. However, to date, there is no study reported the association between ALOX5AP polymorphism and lipids and tHcy profile. In our study, there was no association of ALOX5AP genotypes with lipid profiles or total homocysteine levels. But levels of TC, LDL, HDL, and tHcy were obviously different in men and women, indicating that their levels were influenced by sex, life-style, and other factors. As reported previously, many factors significantly increased risk of hyperhomocysteinemia, such as increased age, male sex, family history of stroke, in addition to deficiencies of serum folate and vitamin B12, and elevated serum creatinine [36]. So we speculated that ALOX5AP polymorphisms may contribute little to lipids profile and tHcy level relative to environmental factors in ischemic stroke patients in our region. Ischemic stroke is a complex clinical syndrome with different etiologies and risk factors. Our current data demonstrated that ALOX5AP rs4073259 was not associated with ischemic stroke risk in the northeastern Chinese Han population although TC, LDL, HDL, and tHcy were obviously different in men and women. Undoubtedly, further studies of different ethnic populations with a larger sample size and brain CT or MRI scan in all the subjects will provide insightful information regarding this SNP for association with IS risk.

Conflicts of interests/disclosures None declared.

Acknowledgment The study was supported in part by a grant form the Natural Science Foundation of Heilongjiang Province (H201331).

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