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Association of SAA gene polymorphism with ischemic stroke in northern Chinese Han population
Accepted Manuscript Association of SAA gene polymorphism with ischemic stroke in northern Chinese Han population
Jie Zhao, Xiangyu Piao, Yue Wu, Ping Xu, Zhiyi He PII: DOI: Reference:
S0022-510X(17)30446-X doi: 10.1016/j.jns.2017.07.012 JNS 15436
To appear in:
Journal of the Neurological Sciences
Received date: Revised date: Accepted date:
6 February 2017 15 June 2017 7 July 2017
Please cite this article as: Jie Zhao, Xiangyu Piao, Yue Wu, Ping Xu, Zhiyi He , Association of SAA gene polymorphism with ischemic stroke in northern Chinese Han population. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Jns(2017), doi: 10.1016/j.jns.2017.07.012
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ACCEPTED MANUSCRIPT Association of SAA Gene Polymorphism with Ischemic Stroke in Northern Chinese Han Population Jie Zhao1,4, Xiangyu Piao1, Yue Wu2, Ping Xu3, Zhiyi He4＊ 1 Department of Neurology, Affiliated Zhongshan Hospital of
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Dalian University, Dalian 116001, China
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Dalian University, Dalian 116001, China
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2 Department of Orthopedics, Affiliated Zhongshan Hospital of
3 Department of Neurology, The Fifth People’s Hospital of Dalian,
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Dalian 116021, China
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4 Department of Neurology, The First Affiliated Hospital of China
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Medical University, Shenyang 110001, China
* Corresponding author
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Tel: +86 024 83282515
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E-mail: [email protected](H. Zhiyi)
ACCEPTED MANUSCRIPT Abstract
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stroke
Serum amyloid A protein; Single nucleotide polymorphism; Ischemic
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Key words
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Background: Serum amyloid A protein (SAA) is known as an inflammatory factor and an apolipoprotein that can replace apolipoprotein A-I/II components as the major apolipoprotein of high-density lipoprotein (HDL), which is related to atherosclerosis. The present study is aimed to evaluate whether the SAA gene polymorphism is involved in ischemic stroke in northern Chinese Han population. Methods: In a case-control study, the participants included 396 patients (239 males, 157 females) with ischemic stroke and 360 healthy subjects (211 males, 149 females). The rs12218 polymorphism of the SAA gene was analyzed by polymerase chain reaction and restriction fragment length polymorphism, while the rs2468844 polymorphism of the SAA gene was analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Results: The frequencies of the CC genotype and the C allele of rs12218 were higher in participants with ischemic stroke than in the control group (P=0.020 in males, P=0.001 in large-artery atherosclerosis group，LAA). The frequencies of the AG genotype and the G allele of rs2468844 were higher in participants with ischemic stroke than in the control group (P=0.040 in males, P=0.011 in large-artery atherosclerosis group). Multiple logistic regression analysis revealed the significance of the rs12218 in males and in large-artery atherosclerosis group after adjustment for confounding factors. Conclusion: The rs12218 polymorphism of the SAA gene was associated with ischemic stroke in males and in patients with large-artery atherosclerosis group in northern Chinese Han population.
ACCEPTED MANUSCRIPT 1. Introduction
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Ischemic stroke (IS) is a heterogeneous multifactorial disease that can be affected by both genetic and conventional risk factors [1,2]. The development of IS has been attributed to the interaction of multiple factors, including genetic variants, chronic diseases, risk behaviors, and inflammation [3-7]. Inflammation is a key process in the pathogenesis of atherosclerosis. Accumulated evidences suggest that several environmental risk factors of IS (e.g., smoking, obesity and alcohol) may work through promoting inflammation. Moreover, many epidemiological studies have confirmed that levels of high-density lipoprotein (HDL) have a strong inverse relationship with atherosclerosis and IS. In general, a low level of HDL in plasma is accepted as being a strong and independent risk factor for the development of premature atherosclerosis.
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Serum amyloid A (SAA) is a sensitive marker of the acute inflammatory state and its plasma concentrations markedly increase in response to infection, trauma or stress [8]. The small apolipoprotein SAA, the precursor to amyloid protein A, is required for the amyloidosis disease process and has been reported as functional in tissue factor expression in endothelial cells [9], lipid metabolism and transport, chemotaxis and regulation of the inflammatory process [8]. During the acute phase response, SAA proteins associate with HDL, displacing its apolipoprotein A-I/II components [10]. SAA is primarily synthesized in the liver by activated monocytes and macrophages [11]. Sustained high expression of SAA may contribute to atherogenesis through its interference with the cholesterol transport and anti-oxidant functions of HDL [8,11]. Elevated SAA level is associated with increased progression of atherosclerosis [12,13] and an increased risk of IS events [14,15]. SAA is not only one of the acute phase proteins, but also a kind of apolipoprotein. Studies [16] have shown that in vivo concentrations of SAA in plasma can be dramatically increased (up to 1000-fold) during acute inflammatory conditions [17]. As an apolipoprotein, SAA is associated with HDL and during inflammation can contribute up to 80% of its apoprotein composition [17]. Many studies have demonstrated that sustained high expression of SAA may contribute to atherogenesis [12,13] and that an elevated concentration of SAA is associated with an increased risk of atherosclerosis [14]. In addition, recently study has reported that beside combination with HDL, SAA was also combined with low density lipoprotein(LDL) in chronical inflammatory respond. SAA has been reported as able to cut down the stability of atherosclerosis plaque. In addition, SAA is secreted into plasma, where it associates primarily with HDL particles but also with very-low-density lipoprotein (VLDL) particles [18]. SAA has been shown to mediate the binding of HDL to differentiated macrophages and endothelial cells [19] and to impair the ability of HDL to promote cholesterol efflux from macrophages [20]. However, the relationships between SAA gene polymorphisms and IS remain illusive. The human SAA gene located on the short arm of chromosome11, band p15.1, contains four related genes, SAA1-4, within a 150 bp region [21]. Only the SAA1 and
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SAA2 genes encode acute-phase SAAs (SAA1 and SAA2 proteins), so a lot of research focuses on them. Recently, Carty et al [22] reported an association of SAA1 and SAA2 gene polymorphisms and carotid intima- media thickness (cIMT), HDL, and CVD. The relationship between the genetic polymorphisms of SAA1/2 and carotid cIMT and HDL level in healthy subjects has been reported. Xie X et al [23] reported an association of SAA1 and SAA2 gene polymorphisms and carotid cIMT. However, the relationship between the genetic polymorphisms of SAA1/2 and IS is not fully understood. Therefore, the purpose of the present case-control study was to determine whether the rs12218 and rs2468844 polymorphism of the SAA gene played a role in IS in Han Chinese population and whether it was different in subtypes.
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2. Materials and methods
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2.1. Subjects
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A total of 756 Chinese Han individuals were recruited from outpatient and inpatient services at the hospitals from 1 September 2010 to 31 August 2015 in Shenyang and Dalian city. The study population comprised 396 patients with IS and 360 unrelated healthy control subjects. All patients with a clinical diagnosis of acute IS were confirmed by computed tomography (CT) and/or magnetic resonance imaging (MRI). AS TOAST (Trial of Org 10172 in Acute Stroke) grouping method, ischemic stroke included the large vessels group (large-artery atherosclerosis group, LAA) and the small vessels group (small-artery occlusion group, SAO). Patients who had any history or occurrence of hemorrhagic stroke, cardiovascular disease, cancer, autoimmune disease, or chronic inflammation were excluded from this study. Unrelated healthy controls were matched with patients according to geographic area, ethnic origin, gender, and age and had no clinical history of stroke or cerebrovascular disease, although some had vascular risk factors, such as hypertension, hypercholesterolemia, and diabetes mellitus (DM). Control individuals reporting a history of cardiovascular events other than stroke were eligible. The case and control individuals were interviewed by investigators using a structured questionnaire. Experimental protocols were approved by the Ethics Committee of both hospitals involved in the study. In addition to neurological history, history of hypertension, DM, and the following vascular risk factors were recorded: a history of smoking, alcohol consumption, and body-mass index (Table 1).
2.2. Polymorphism genotyping Human genomic DNA was extracted from EDTA-treated venous blood samples according to the instruction of genomic DNA purification kit (Wizard Genomic DNA purification kit; Promega, USA). DNA samples were stored at −20 °C. And its purity and concentration were analyzed and detected through a spectrophotometer. The sequence of SAA was obtained from GenBank (NC-000011.9). The rs12218 polymorphism of the SAA gene was analyzed by polymerase chain reaction(PCR) and restriction fragment length polymorphism(RFLP), while the rs2468844
ACCEPTED MANUSCRIPT polymorphism of the SAA gene was analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS).
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For rs12218, primers were designed using Primer Premier 5.0. Sequences were as follows: sense, 5′- AAC AGG GAG AAT GGG AGG GTG GG -3′, and antisense, 5′GCA GGT CGG AAG TGA TTG GGG TC-3′, with an amplified fragment length of 193bp. Reactions were carried out in a 25-μl reaction volume containing 1μl DNA, 0.5 μmol/l of each primer, 2.5 mM each dNTP, 2.5 μl 10X PCR buffer, and 0.625 U Taq DNA polymerase (Takara, China). Amplification was carried out as follows: 94°C for 5 min, followed by 35 cycles of 94°C for 30 s, 58°C for 30 s, and 72°C for 1 min, with a final extension step at 72°C for 10 min. Products were digested overnight at 37°C with the restriction enzyme Bg1I (Takara, China) according to the manufacturer's recommendation. The CC homozygote produced two fragments (130 bp and 63 bp), the CT heterozygote produced three fragments (193 bp, 130 bp, and 63 bp), and the 193-bp fragment remained intact in TT homozygotes. The restriction-digest products were analyzed by electrophoresis on 2.0% agarose and genefounder™ (Bio-V, Xiamen, China) staining.
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For rs2468844, primers were designed using the Sequenom software package. The sequences were as follows: sense, 5′- ACG TTG GAT GTT TAT TGG CAG CCT GAT CGG -3′, antisense, 5′- ACG TTG GAT GGC CAG AGA GAA TAT CCA GAG-3′. In addition, the single-base extension primer sequence was 5′- CGA GTC CTC CGC ACC A-3′. Reactions were performed in a total volume of 5 µL, containing 1 µL DNA, 0.5 µL HotStar Taq polymerase (5 U/µL), 0.325 µL MgCl2 (25 mmol/L), 0.625 µL 10X PCR buffer, 1 µL dNTP mix (2.5 mmol/L each), 0.95 µL H2 O, and 1 µL designed primers at their optimized concentrations (all from Sequenom Inc.). Cycling conditions were as follows: 94°C for 15 s, followed by 45 cycles of 94°C for 20 s, 56°C for 30 s, and 72°C for 1 min, with a final extension step at 72°C for 3 min. Samples were kept at 4°C until further analysis. After PCR, the products were treated with shrimp alkaline phosphatase (SAP) to remove excess dNTPs in a reaction containing 0.3 µL SAP (1.7 U/µL), 0.17 µL 10X SAP buffer, and 1.53 µL H2 O (all from Sequenom Inc.). The reaction conditions were as follows: 37°C for 40 min, followed by 85°C for 5 min. Samples were kept at 12°C until further analysis. The PCR products were then used as templates for the primer extension reactions using the iPlex1 Gold reagent kit (Sequenom Inc.). The extension reactions were performed in a final volume of 9 µL, containing 0.2 µL 10X iPlex buffer, 0.2 µL iPlex termination mix, 0.041 µL iPlex enzyme, 0.619 µL H2 O, and 0.940 µL extension primers at their optimized concentrations. The extension reaction conditions were as follows: 94°C for 30 s, followed by 40 cycles of 94°C for 5 s, 5 cycles of 52°C for 5 s and 80°C for 5 s, and a final step of 72°C for 3 min. The final nucleotide extension products were treated with a cationic exchange resin for 30 min to remove salts. All reactions, including the PCR amplification, SAP treatment, and primer extension, were performed in 384-well microtiter plates (Sequenom Inc.). The PCR amplification and primer extension reaction were performed on a GeneAmp® PCR
ACCEPTED MANUSCRIPT System 9700 Dual 384-well Sample Block Module and non-template controls were included in every plate to confirm that there was no contamination. The products were spotted onto the MassARRAY SpectroCHIP with an auto-spot arm (Sequenom Inc.) and air-dried. The target plate was then inserted into the MALDI-TOF mass spectrometer of the MassARRAY Compact System (Sequenom Inc.) and analysis detection was performed.
2.3. Statistical analysis
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Statistical analyses were performed with SPSS 13.0. Data are presented as the mean±SD or percent frequency. Allele frequencies were calculated from the genotypes of all subjects. The alleles and genotype frequencies of SAA between the patients and controls were compared using a chi-square test or Fisher's exact test. The Hardy–Weinberg equilibrium (HWE) was assessed by HWE software. Risk factors were screened by the Student's t test or chi-square test. The relationship between different genotypes of SAA and ischemic stroke was evaluated by logistic regression analysis after adjusting for confounding covariates. A P-value < 0.05 was considered statistically significant.
3. Results
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The characteristics of the patients and controls are shown in Table 1. Comparing patients to controls, there was no significant difference in age, gender, or body-mass index (BMI). For all participants, significantly higher values were obtained for IS patients than controls for prevalence of hypertension, diabetes mellitus, smoking and alcohol consumption, systolic blood pressure (SBP), diastolic blood pressure (DBP), blood glucose, total plasma cholesterol, triglycerides, and plasma LDL. There was no significant difference with respect to mean plasma HDL between patients and controls. All genotype distributions in both groups were in HWE. For rs12218, P was 0.266 and 0.681 for the patient and control groups, respectively, while for rs2468844, P was 0.116 and 0.378, respectively.
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The distribution of the SAA polymorphisms and the allelic frequency in patients and controls are given in Table 2-6. In male and LAA subtypes, there was a significant difference in the allelic distribution between patients and control subjects. Multivariate logistic regression analysis was used to evaluate the relationships between the polymorphism and ischemic stroke in males and those in the LAA subtype. After adjustment for confounding variables, the CC genotype and C allele of rs12218 was still significantly associated with an increased risk of IS compared with the TT genotype and T in males (OR=2.66, P=0.036) and LAA (OR=2.34, P=0.044). The AG genotype and G allele of rs2468844 was no statistically significant difference in either the genotypic distribution or allelic frequency between the patients and controls. Analysis of the interaction of genotypes is shown in Table 2-6.
4. Discussion This study investigated the association between SAA polymorphisms, rs12218 and
ACCEPTED MANUSCRIPT rs2468844, and ischemic stroke in Northern Chinese Han population. In this study, we found that the genotypic and allelic frequencies of rs12218 was associated with IS in male and LAA subtypes, but not in female or SAO subtypes. However, the rs2468844 has no significant difference between the patients and controls after adjustment for confounding variables.
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Recently, the SNPs of SAA1 and SAA2 has been found associated with coronary artery disease and myocardial infarction, there is no report about whether these SNPs can also be associated with risk for IS in Han Chinese population. In previous studies, we have suggested the gene SNPs, such as PDE4D，IL-1, CYP4F2, adiponectin , apolipoprotein M ,RANTES and ALOX15,are associated with increased risk of IS in a Chinese Han population [24-29].In this study, we explored the potential association of SAA SNPs with IS in northern Chinese Han population.
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Previous studies have reported that elevated basal levels of SAA were shown to be predictive of future vascular events [14]. Yamada et al. [30] reported that the SAA1 allele influences the plasma concentration of SAA in the Japanese population and person taking along with the SAA1.5 allele have a higher plasma concentration of SAA than those lacking this allele. Carty et al. [22] reported that SAA2 SNPs were associated with increased IMT in African-American subjects, but this association was not observed in European-American subjects. Xie X et al. [23] considered that SAA rs2468844 and rs12218 (interaction P,0.001) and rs2229338 (interaction P = 0.001) may be associated with the serum concentration of HDL and carotid IMT, this result was in accordance with the finding reported by Carty et al. In our study, we found SAA1 (rs12218) was associated with IS in northern Chinese Han population after adjustment for age, sex, and other factors. However, the mechanisms which may link SAA1/2 genetic polymorphisms to IS are largely unknown. Carty et al. [22] indicated that variation in SAA1/2 could lead to altered binding affinity of the SAA proteins for HDL and that the association between SAA1/2 genetic variants and CVD may be modified by HDL. Although neither the study by Carty et al. nor the study by Xie X et al employed the functional analysis of variation in SAA1/2 with respect to the relationship between SAA1/2 and cIMT. Whether both of those two polymorphisms affect the stability or binding of the protein is still unknown. In our study, according to TOAST typing method, patients have been typed two subtypes, the large vessels group (large-artery atherosclerosis group) and the small vessels group (small-artery occlusion group). LAA associated with atherosclerosis is thrombo-ischemic stroke in large and moderately large artery. SAO is caused by small cerebro-artery hyalinization, vascular sclerosis and fiber necrosis because of hypertension. Our findings suggested that SAA rs12218 may be a significantly higher risk factor for IS and rs12218 may weakly associated with male gender and LAA. The results are in concordance with the genetic factors play a greater role in atherosclerosis from IS. To our knowledge this is the first study to investigate common allelic variants in the SAA genes and their association with IS. Inflammation
ACCEPTED MANUSCRIPT is an important contributor to atherosclerosis and gene variants mediating inflammation are of interest. We hope that future research on SAA gene may contribute to its role in atherosclerosis and IS.
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In summary, our study demonstrated that the polymorphisms in SAA, rs12218 may be associated with susceptibility of IS, which is independent to the other common ischemic stroke risk factors in northern Chinese Han population. However, it is recognized that the value of this study was limited by a relative small sample size and only a few SNPs in each gene studied. This study was undertaken on a relatively older population and hence the results may not be applicable to a younger cohort. Accordingly, we are interested in further studying the association of these polymorphisms with IS in a big Chinese Han population with multiple SNPs in each gene to verify these findings. After perspective studies within a large population to confirm the significance of these SNPs, they can be used as the prediction of IS in a Chinese Han population conceivably.
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Funding for the study
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This study was supported by a grant from the National Natural Science Foundation of China (81070913,81501753).
Conflicts of interest
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The authors declare no conflicts of interest.
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thickness in healthy Han Chinese subjects: the Cardiovascular Risk Survey. Plos One 2010;5(11): e13997. 24.Li N et al. Association of PDE4D and IL-1 gene polymorphism with ischemic stroke in a Han Chinese population. Brain Res Bull 2010;81(1):38-42. 25.Deng S et al. CYP4F2 gene V433M polymorphism is associated with ischemic stroke in the male Northern Chinese Han population. Prog Neuropsychopharmacol Biol Psychiatry 2010;34(4):664-668. 26.Liu F et al. Association of adiponectin gene polymorphisms with the risk of ischemic stroke in a Chinese Han population. Mol Biol Rep 2011;38(3):1983-1988. 27.Zhao D et al. Association of apolipoprotein M gene polymorphisms with ischemic stroke in a Han Chinese population. J Mol Neurosci 2011;43(3):370-375. 28.Qin X et al. The RANTES gene promoter polymorphisms are associated with the risk of atherothrombotic cerebral infarction in Northern Han Chinese. Clinica Chimica Acta 2011; 412(11-12):1112-1115. 29.Zhao J et al. Association of ALOX15 gene polymorphism with ischemic stroke in Northern
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Table 1 Characteristics of the participants between the ischemic stroke and control group Total Controls (n=360)
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Age (year)
63.4±9.0
62.6±8.7
0.210
Gender (male/female) BMI (kg/m2 )
239/157 24.3±2.15
211/149 24.0±2.08
0.626 0.057
Hypertension (%) Diabet esmellits (%)
234 (59.1) 106 (26.8)
112 (31.1) 60 (16.7)
<0.001 <0.001
Smoking (%)
103 (26.0)
41 (11.4)
<0.001
Alcohol drinker (%) SBP (mmHg)
97 (24.5) 143±18
37 (10.3) 132±15
<0.001 <0.001
DBP (mmHg) Blood glucose (mmol/l)
84±10 6.42±2.17
79±9 5.72±1.14
<0.001 <0.001
Total cholesterol (mg/dl) Triglycerides (mmol/l)
4.76±1.05 1.59±1.03
4.44±0.87 1.32±0.65
<0.001 <0.001
HDL (mmol/l) LDL (mmol/l)
1.14±0.31 3.19±0.97
1.15±0.27 2.83±0.76
0.494 <0.001
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Cases (n=396)
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Data are presented as the mean±SD or percent frequency. n: Number; SD: standard deviation; BMI, body-mass index; SBP, systolic blood pressure; DBP, diastolic blood pressure; HDL-C,
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high-density lipoprotein cholesterol; LDL-C, low-density lipoprotein cholesterol. P values from Student's t-test or χ2. Table 2 Genotype and allele distributions for the patients with ischemic stroke and the control group Total
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Cases
n (%)
rs12218
Genotype
Allele rs2468844 Genotype Allele
Controls n (%)
Single analysis P
OR (95%CI)
211(53.3) 150(37.9)
223(61.9) 119(33.1)
CC T
35(8.8) 572(72.2)
18(5.0) 565(78.5)
0.018
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220(27.8)
155(21.5)
0.005
AA
338(85.4)
328(91.1)
AG
58(14.6)
32(8.9)
A
734(92.7)
688(95.6)
0.066
regression analysis P OR (95%CI) 0.045
0.023
TT CT
Multiple logistic
Reference 1.33(0.98-1.81)
Reference 0.126
1.25(0.91-1.79)
2.06(1.13-3.74) Reference
0.044
1.64(1.03-3.29)
1.40(1.11-1.78)
0.048
Reference
Reference 0.016
1.76(1.11-2.78) Reference
1.27(1.01-1.68)
Reference 0.241
1.37(0.81-2.31)
Reference
1.34(0.81-2.22) G 58(7.3) 32(4.4) 0.019 1.70(1.09-2.65) 0.259 Data are adjusted for hypertension, diabetes mellitus, smoking and alcohol consumption, systolic
ACCEPTED MANUSCRIPT blood pressure (SBP), diastolic blood pressure (DBP), blood glucose, total plasma cholesterol, triglycerides, and plasma LDL; OR, odds ratio; CI, credibility interval.
Table 3 Genotype and allele distributions for the patients with the male and the control group males Case n (%)
rs2468844 Genotype
P
OR (95%CI)
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340(71.1) 138(28.9)
332(78.7) 90(21.3)
AA AG
202(84.5) 37(15.5)
192(91.0) 19(9.0)
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131(62.1) 70(33.2) 10(4.7)
0.141 0.020
Reference 1.35(0.91-2.00) 2.52(1.16-5.47)
0.010
Reference 1.50(1.1-2.03)
0.040
Reference 1.85(1.03-3.33)
1.47(0.91-2.37)
0.036
2.66(1.07-6.62)
0.010
Reference 1.57(1.09-2.26)
Reference 0.156
1.62(0.83-3.15)
A 441(92.3) 403(95.5) Reference Reference 1.56(0.82-2.96) G 37(7.7) 19(4.5) 0.047 1.78(1.01-3.14) 0.170 Data are adjusted for hypertension, diabetes mellitus, smoking and alcohol consumption, systolic
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Reference
0.115
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125(52.3) 90(37.7) 24(10.0)
regression analysis P OR (95%CI) 0.049
0.039 TT CT CC
Multiple logistic
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n (%)
Single analysis
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rs12218 Genotype
Control
blood pressure (SBP), diastolic blood pressure (DBP), blood glucose, total plasma cholesterol,
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triglycerides, and plasma LDL; OR, odds ratio; CI, credibility interval.
Table 4 Genotype and allele distributions for the patients with the female and the control group females
n (%) rs12218
Allele
n (%)
TT CT
86(54.8) 60(38.2)
92(61.7) 49(32.9)
CC T C
11(7) 232(73.9) 82(26.1)
8(5.4) 233(78.2) 65(21.8)
AA
136(86.6)
136(91.3)
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Genotype
Control
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Case
Single analysis P
OR (95%CI)
0.770
0.455 0.269 0.429 0.213
Multiple logistic regression analysis P OR (95%CI)
Reference 1.31(0.81-2.11) 1.47(0.56-3.83) Reference 1.27(0.87-1.84)
Reference 0.999
1.00(0.57-1.77)
0.477
0.64(0.19-2.18)
Reference 0.660
0.9(0.58-1.42)
rs2468844 Genotype
AG 21(13.4) 13(8.7) 0.199 Allele A 293(93.3) 285(95.6) G 21(6.7) 13(4.4) 0.213 Data are adjusted for hypertension, diabetes mellitus,
Reference
Reference
1.04(0.42-2.55) 1.62(0.78-3.36) 0.933 Reference Reference 0.940 1.04(0.44-2.47) 1.57(0.77-3.2) smoking and alcohol consumption, systolic
blood pressure (SBP), diastolic blood pressure (DBP), blood glucose, total plasma cholesterol, triglycerides, and plasma LDL; OR, odds ratio; CI, credibility interval.
ACCEPTED MANUSCRIPT Table 5 Genotype and allele distributions for the patients with the LAA and the control group LAA
Allele
Control
n (%)
n (%)
Single analysis P
OR (95%CI)
regression analysis OR (95%CI)
TT
85(48)
223(61.9)
CT
70(39.5)
119(33.1)
0.028
Reference 1.54(1.05-2.27)
0.248
1.32(0.82-2.11)
CC
22(12.4)
18(5)
0.001
3.21(1.64-6.27)
0.044
2.34(1.02-5.33)
T
240(67.8)
565(78.5)
C
114(32.2)
155(21.5)
AA AG
148(83.6) 29(16.4)
328(91.1) 32(8.9)
A
325(91.8)
688(95.6)
Reference
Reference
0.000
1.73(1.30-2.30)
0.011
Reference 2.01(1.17-3.44)
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0.036
Reference 1.45(1.02-2.06)
Reference
0.195
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Allele
P
0.046
0.001
rs2468844 Genotype
Multiple logistic
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rs12218 Genotype
Case
Reference
1.54(0.8-2.96)
Reference
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1.49(0.79-2.78) G 29(8.2) 32(4.4) 0.014 1.92(1.14-3.23) 0.214 Data are adjusted for hypertension, diabetes mellitus, smoking and alcohol consumption, systolic
blood pressure (SBP), diastolic blood pressure (DBP), blood glucose, total plasma cholesterol,
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triglycerides, and plasma LDL; OR, odds ratio; CI, credibility interval.
Table 6 Genotype and allele distributions for the patients with the SAO and the control group SAO
rs12218 TT
126(57.5)
223(61.9)
CT CC
80(36.5) 13(5.9)
119(33.1) 18(5)
T C
332(75.8) 106(24.2)
565(78.5) 155(21.5)
rs2468844 Genotype
AA
190(86.8)
328(91.1)
Allele
AG A
29(13.2) 409(93.4)
32(8.9) 688(95.6)
Allele
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Genotype
n (%)
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n (%)
Control
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Case
Single analysis P
OR (95%CI)
Multiple logistic regression analysis P 0.739
0.566 Reference 0.341 0.519
1.19(0.83-1.7) 1.28(0.61-2.7)
0.291
Reference 1.16(0.88-1.54)
Reference 0.478
1.16(0.77-1.75)
0.660
1.21(0.51-2.87)
Reference 0.448
Reference 0.100
OR (95%CI)
1.56(0.92-2.67) Reference
1.13(0.82-1.57)
Reference 0.691
1.13(0.61-2.11)
Reference
1.12(0.62-2.05) G 29(6.6) 32(4.4) 0.110 1.52(0.91-2.56) 0.702 Data are adjusted for hypertension, diabetes mellitus, smoking and alcohol consumption, systolic
blood pressure (SBP), diastolic blood pressure (DBP), blood glucose, total plasma cholesterol, triglycerides, and plasma LDL; OR, odds ratio; CI, credibility interval.
ACCEPTED MANUSCRIPT Highlights
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Our study focused on Northern Chinese Han population. The SAA gene polymorphisms were associated with susceptibility to ischemic stroke. The SNPs of SAA gene had a significant in males. The SNPs of SAA gene had a significant in atherosclerosis ischemic stroke subtype.
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Jie Zhao, Xiangyu Piao, Yue Wu, Ping Xu, Zhiyi He PII: DOI: Reference:
S0022-510X(17)30446-X doi: 10.1016/j.jns.2017.07.012 JNS 15436
To appear in:
Journal of the Neurological Sciences
Received date: Revised date: Accepted date:
6 February 2017 15 June 2017 7 July 2017
Please cite this article as: Jie Zhao, Xiangyu Piao, Yue Wu, Ping Xu, Zhiyi He , Association of SAA gene polymorphism with ischemic stroke in northern Chinese Han population. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Jns(2017), doi: 10.1016/j.jns.2017.07.012
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ACCEPTED MANUSCRIPT Association of SAA Gene Polymorphism with Ischemic Stroke in Northern Chinese Han Population Jie Zhao1,4, Xiangyu Piao1, Yue Wu2, Ping Xu3, Zhiyi He4＊ 1 Department of Neurology, Affiliated Zhongshan Hospital of
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Dalian University, Dalian 116001, China
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Dalian University, Dalian 116001, China
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2 Department of Orthopedics, Affiliated Zhongshan Hospital of
3 Department of Neurology, The Fifth People’s Hospital of Dalian,
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Dalian 116021, China
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4 Department of Neurology, The First Affiliated Hospital of China
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Medical University, Shenyang 110001, China
* Corresponding author
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Tel: +86 024 83282515
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E-mail: [email protected](H. Zhiyi)
ACCEPTED MANUSCRIPT Abstract
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stroke
Serum amyloid A protein; Single nucleotide polymorphism; Ischemic
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Key words
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Background: Serum amyloid A protein (SAA) is known as an inflammatory factor and an apolipoprotein that can replace apolipoprotein A-I/II components as the major apolipoprotein of high-density lipoprotein (HDL), which is related to atherosclerosis. The present study is aimed to evaluate whether the SAA gene polymorphism is involved in ischemic stroke in northern Chinese Han population. Methods: In a case-control study, the participants included 396 patients (239 males, 157 females) with ischemic stroke and 360 healthy subjects (211 males, 149 females). The rs12218 polymorphism of the SAA gene was analyzed by polymerase chain reaction and restriction fragment length polymorphism, while the rs2468844 polymorphism of the SAA gene was analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Results: The frequencies of the CC genotype and the C allele of rs12218 were higher in participants with ischemic stroke than in the control group (P=0.020 in males, P=0.001 in large-artery atherosclerosis group，LAA). The frequencies of the AG genotype and the G allele of rs2468844 were higher in participants with ischemic stroke than in the control group (P=0.040 in males, P=0.011 in large-artery atherosclerosis group). Multiple logistic regression analysis revealed the significance of the rs12218 in males and in large-artery atherosclerosis group after adjustment for confounding factors. Conclusion: The rs12218 polymorphism of the SAA gene was associated with ischemic stroke in males and in patients with large-artery atherosclerosis group in northern Chinese Han population.
ACCEPTED MANUSCRIPT 1. Introduction
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Ischemic stroke (IS) is a heterogeneous multifactorial disease that can be affected by both genetic and conventional risk factors [1,2]. The development of IS has been attributed to the interaction of multiple factors, including genetic variants, chronic diseases, risk behaviors, and inflammation [3-7]. Inflammation is a key process in the pathogenesis of atherosclerosis. Accumulated evidences suggest that several environmental risk factors of IS (e.g., smoking, obesity and alcohol) may work through promoting inflammation. Moreover, many epidemiological studies have confirmed that levels of high-density lipoprotein (HDL) have a strong inverse relationship with atherosclerosis and IS. In general, a low level of HDL in plasma is accepted as being a strong and independent risk factor for the development of premature atherosclerosis.
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Serum amyloid A (SAA) is a sensitive marker of the acute inflammatory state and its plasma concentrations markedly increase in response to infection, trauma or stress [8]. The small apolipoprotein SAA, the precursor to amyloid protein A, is required for the amyloidosis disease process and has been reported as functional in tissue factor expression in endothelial cells [9], lipid metabolism and transport, chemotaxis and regulation of the inflammatory process [8]. During the acute phase response, SAA proteins associate with HDL, displacing its apolipoprotein A-I/II components [10]. SAA is primarily synthesized in the liver by activated monocytes and macrophages [11]. Sustained high expression of SAA may contribute to atherogenesis through its interference with the cholesterol transport and anti-oxidant functions of HDL [8,11]. Elevated SAA level is associated with increased progression of atherosclerosis [12,13] and an increased risk of IS events [14,15]. SAA is not only one of the acute phase proteins, but also a kind of apolipoprotein. Studies [16] have shown that in vivo concentrations of SAA in plasma can be dramatically increased (up to 1000-fold) during acute inflammatory conditions [17]. As an apolipoprotein, SAA is associated with HDL and during inflammation can contribute up to 80% of its apoprotein composition [17]. Many studies have demonstrated that sustained high expression of SAA may contribute to atherogenesis [12,13] and that an elevated concentration of SAA is associated with an increased risk of atherosclerosis [14]. In addition, recently study has reported that beside combination with HDL, SAA was also combined with low density lipoprotein(LDL) in chronical inflammatory respond. SAA has been reported as able to cut down the stability of atherosclerosis plaque. In addition, SAA is secreted into plasma, where it associates primarily with HDL particles but also with very-low-density lipoprotein (VLDL) particles [18]. SAA has been shown to mediate the binding of HDL to differentiated macrophages and endothelial cells [19] and to impair the ability of HDL to promote cholesterol efflux from macrophages [20]. However, the relationships between SAA gene polymorphisms and IS remain illusive. The human SAA gene located on the short arm of chromosome11, band p15.1, contains four related genes, SAA1-4, within a 150 bp region [21]. Only the SAA1 and
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SAA2 genes encode acute-phase SAAs (SAA1 and SAA2 proteins), so a lot of research focuses on them. Recently, Carty et al [22] reported an association of SAA1 and SAA2 gene polymorphisms and carotid intima- media thickness (cIMT), HDL, and CVD. The relationship between the genetic polymorphisms of SAA1/2 and carotid cIMT and HDL level in healthy subjects has been reported. Xie X et al [23] reported an association of SAA1 and SAA2 gene polymorphisms and carotid cIMT. However, the relationship between the genetic polymorphisms of SAA1/2 and IS is not fully understood. Therefore, the purpose of the present case-control study was to determine whether the rs12218 and rs2468844 polymorphism of the SAA gene played a role in IS in Han Chinese population and whether it was different in subtypes.
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2. Materials and methods
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2.1. Subjects
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A total of 756 Chinese Han individuals were recruited from outpatient and inpatient services at the hospitals from 1 September 2010 to 31 August 2015 in Shenyang and Dalian city. The study population comprised 396 patients with IS and 360 unrelated healthy control subjects. All patients with a clinical diagnosis of acute IS were confirmed by computed tomography (CT) and/or magnetic resonance imaging (MRI). AS TOAST (Trial of Org 10172 in Acute Stroke) grouping method, ischemic stroke included the large vessels group (large-artery atherosclerosis group, LAA) and the small vessels group (small-artery occlusion group, SAO). Patients who had any history or occurrence of hemorrhagic stroke, cardiovascular disease, cancer, autoimmune disease, or chronic inflammation were excluded from this study. Unrelated healthy controls were matched with patients according to geographic area, ethnic origin, gender, and age and had no clinical history of stroke or cerebrovascular disease, although some had vascular risk factors, such as hypertension, hypercholesterolemia, and diabetes mellitus (DM). Control individuals reporting a history of cardiovascular events other than stroke were eligible. The case and control individuals were interviewed by investigators using a structured questionnaire. Experimental protocols were approved by the Ethics Committee of both hospitals involved in the study. In addition to neurological history, history of hypertension, DM, and the following vascular risk factors were recorded: a history of smoking, alcohol consumption, and body-mass index (Table 1).
2.2. Polymorphism genotyping Human genomic DNA was extracted from EDTA-treated venous blood samples according to the instruction of genomic DNA purification kit (Wizard Genomic DNA purification kit; Promega, USA). DNA samples were stored at −20 °C. And its purity and concentration were analyzed and detected through a spectrophotometer. The sequence of SAA was obtained from GenBank (NC-000011.9). The rs12218 polymorphism of the SAA gene was analyzed by polymerase chain reaction(PCR) and restriction fragment length polymorphism(RFLP), while the rs2468844
ACCEPTED MANUSCRIPT polymorphism of the SAA gene was analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS).
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For rs12218, primers were designed using Primer Premier 5.0. Sequences were as follows: sense, 5′- AAC AGG GAG AAT GGG AGG GTG GG -3′, and antisense, 5′GCA GGT CGG AAG TGA TTG GGG TC-3′, with an amplified fragment length of 193bp. Reactions were carried out in a 25-μl reaction volume containing 1μl DNA, 0.5 μmol/l of each primer, 2.5 mM each dNTP, 2.5 μl 10X PCR buffer, and 0.625 U Taq DNA polymerase (Takara, China). Amplification was carried out as follows: 94°C for 5 min, followed by 35 cycles of 94°C for 30 s, 58°C for 30 s, and 72°C for 1 min, with a final extension step at 72°C for 10 min. Products were digested overnight at 37°C with the restriction enzyme Bg1I (Takara, China) according to the manufacturer's recommendation. The CC homozygote produced two fragments (130 bp and 63 bp), the CT heterozygote produced three fragments (193 bp, 130 bp, and 63 bp), and the 193-bp fragment remained intact in TT homozygotes. The restriction-digest products were analyzed by electrophoresis on 2.0% agarose and genefounder™ (Bio-V, Xiamen, China) staining.
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For rs2468844, primers were designed using the Sequenom software package. The sequences were as follows: sense, 5′- ACG TTG GAT GTT TAT TGG CAG CCT GAT CGG -3′, antisense, 5′- ACG TTG GAT GGC CAG AGA GAA TAT CCA GAG-3′. In addition, the single-base extension primer sequence was 5′- CGA GTC CTC CGC ACC A-3′. Reactions were performed in a total volume of 5 µL, containing 1 µL DNA, 0.5 µL HotStar Taq polymerase (5 U/µL), 0.325 µL MgCl2 (25 mmol/L), 0.625 µL 10X PCR buffer, 1 µL dNTP mix (2.5 mmol/L each), 0.95 µL H2 O, and 1 µL designed primers at their optimized concentrations (all from Sequenom Inc.). Cycling conditions were as follows: 94°C for 15 s, followed by 45 cycles of 94°C for 20 s, 56°C for 30 s, and 72°C for 1 min, with a final extension step at 72°C for 3 min. Samples were kept at 4°C until further analysis. After PCR, the products were treated with shrimp alkaline phosphatase (SAP) to remove excess dNTPs in a reaction containing 0.3 µL SAP (1.7 U/µL), 0.17 µL 10X SAP buffer, and 1.53 µL H2 O (all from Sequenom Inc.). The reaction conditions were as follows: 37°C for 40 min, followed by 85°C for 5 min. Samples were kept at 12°C until further analysis. The PCR products were then used as templates for the primer extension reactions using the iPlex1 Gold reagent kit (Sequenom Inc.). The extension reactions were performed in a final volume of 9 µL, containing 0.2 µL 10X iPlex buffer, 0.2 µL iPlex termination mix, 0.041 µL iPlex enzyme, 0.619 µL H2 O, and 0.940 µL extension primers at their optimized concentrations. The extension reaction conditions were as follows: 94°C for 30 s, followed by 40 cycles of 94°C for 5 s, 5 cycles of 52°C for 5 s and 80°C for 5 s, and a final step of 72°C for 3 min. The final nucleotide extension products were treated with a cationic exchange resin for 30 min to remove salts. All reactions, including the PCR amplification, SAP treatment, and primer extension, were performed in 384-well microtiter plates (Sequenom Inc.). The PCR amplification and primer extension reaction were performed on a GeneAmp® PCR
ACCEPTED MANUSCRIPT System 9700 Dual 384-well Sample Block Module and non-template controls were included in every plate to confirm that there was no contamination. The products were spotted onto the MassARRAY SpectroCHIP with an auto-spot arm (Sequenom Inc.) and air-dried. The target plate was then inserted into the MALDI-TOF mass spectrometer of the MassARRAY Compact System (Sequenom Inc.) and analysis detection was performed.
2.3. Statistical analysis
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Statistical analyses were performed with SPSS 13.0. Data are presented as the mean±SD or percent frequency. Allele frequencies were calculated from the genotypes of all subjects. The alleles and genotype frequencies of SAA between the patients and controls were compared using a chi-square test or Fisher's exact test. The Hardy–Weinberg equilibrium (HWE) was assessed by HWE software. Risk factors were screened by the Student's t test or chi-square test. The relationship between different genotypes of SAA and ischemic stroke was evaluated by logistic regression analysis after adjusting for confounding covariates. A P-value < 0.05 was considered statistically significant.
3. Results
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The characteristics of the patients and controls are shown in Table 1. Comparing patients to controls, there was no significant difference in age, gender, or body-mass index (BMI). For all participants, significantly higher values were obtained for IS patients than controls for prevalence of hypertension, diabetes mellitus, smoking and alcohol consumption, systolic blood pressure (SBP), diastolic blood pressure (DBP), blood glucose, total plasma cholesterol, triglycerides, and plasma LDL. There was no significant difference with respect to mean plasma HDL between patients and controls. All genotype distributions in both groups were in HWE. For rs12218, P was 0.266 and 0.681 for the patient and control groups, respectively, while for rs2468844, P was 0.116 and 0.378, respectively.
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The distribution of the SAA polymorphisms and the allelic frequency in patients and controls are given in Table 2-6. In male and LAA subtypes, there was a significant difference in the allelic distribution between patients and control subjects. Multivariate logistic regression analysis was used to evaluate the relationships between the polymorphism and ischemic stroke in males and those in the LAA subtype. After adjustment for confounding variables, the CC genotype and C allele of rs12218 was still significantly associated with an increased risk of IS compared with the TT genotype and T in males (OR=2.66, P=0.036) and LAA (OR=2.34, P=0.044). The AG genotype and G allele of rs2468844 was no statistically significant difference in either the genotypic distribution or allelic frequency between the patients and controls. Analysis of the interaction of genotypes is shown in Table 2-6.
4. Discussion This study investigated the association between SAA polymorphisms, rs12218 and
ACCEPTED MANUSCRIPT rs2468844, and ischemic stroke in Northern Chinese Han population. In this study, we found that the genotypic and allelic frequencies of rs12218 was associated with IS in male and LAA subtypes, but not in female or SAO subtypes. However, the rs2468844 has no significant difference between the patients and controls after adjustment for confounding variables.
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Recently, the SNPs of SAA1 and SAA2 has been found associated with coronary artery disease and myocardial infarction, there is no report about whether these SNPs can also be associated with risk for IS in Han Chinese population. In previous studies, we have suggested the gene SNPs, such as PDE4D，IL-1, CYP4F2, adiponectin , apolipoprotein M ,RANTES and ALOX15,are associated with increased risk of IS in a Chinese Han population [24-29].In this study, we explored the potential association of SAA SNPs with IS in northern Chinese Han population.
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Previous studies have reported that elevated basal levels of SAA were shown to be predictive of future vascular events [14]. Yamada et al. [30] reported that the SAA1 allele influences the plasma concentration of SAA in the Japanese population and person taking along with the SAA1.5 allele have a higher plasma concentration of SAA than those lacking this allele. Carty et al. [22] reported that SAA2 SNPs were associated with increased IMT in African-American subjects, but this association was not observed in European-American subjects. Xie X et al. [23] considered that SAA rs2468844 and rs12218 (interaction P,0.001) and rs2229338 (interaction P = 0.001) may be associated with the serum concentration of HDL and carotid IMT, this result was in accordance with the finding reported by Carty et al. In our study, we found SAA1 (rs12218) was associated with IS in northern Chinese Han population after adjustment for age, sex, and other factors. However, the mechanisms which may link SAA1/2 genetic polymorphisms to IS are largely unknown. Carty et al. [22] indicated that variation in SAA1/2 could lead to altered binding affinity of the SAA proteins for HDL and that the association between SAA1/2 genetic variants and CVD may be modified by HDL. Although neither the study by Carty et al. nor the study by Xie X et al employed the functional analysis of variation in SAA1/2 with respect to the relationship between SAA1/2 and cIMT. Whether both of those two polymorphisms affect the stability or binding of the protein is still unknown. In our study, according to TOAST typing method, patients have been typed two subtypes, the large vessels group (large-artery atherosclerosis group) and the small vessels group (small-artery occlusion group). LAA associated with atherosclerosis is thrombo-ischemic stroke in large and moderately large artery. SAO is caused by small cerebro-artery hyalinization, vascular sclerosis and fiber necrosis because of hypertension. Our findings suggested that SAA rs12218 may be a significantly higher risk factor for IS and rs12218 may weakly associated with male gender and LAA. The results are in concordance with the genetic factors play a greater role in atherosclerosis from IS. To our knowledge this is the first study to investigate common allelic variants in the SAA genes and their association with IS. Inflammation
ACCEPTED MANUSCRIPT is an important contributor to atherosclerosis and gene variants mediating inflammation are of interest. We hope that future research on SAA gene may contribute to its role in atherosclerosis and IS.
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In summary, our study demonstrated that the polymorphisms in SAA, rs12218 may be associated with susceptibility of IS, which is independent to the other common ischemic stroke risk factors in northern Chinese Han population. However, it is recognized that the value of this study was limited by a relative small sample size and only a few SNPs in each gene studied. This study was undertaken on a relatively older population and hence the results may not be applicable to a younger cohort. Accordingly, we are interested in further studying the association of these polymorphisms with IS in a big Chinese Han population with multiple SNPs in each gene to verify these findings. After perspective studies within a large population to confirm the significance of these SNPs, they can be used as the prediction of IS in a Chinese Han population conceivably.
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Funding for the study
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This study was supported by a grant from the National Natural Science Foundation of China (81070913,81501753).
Conflicts of interest
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The authors declare no conflicts of interest.
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Table 1 Characteristics of the participants between the ischemic stroke and control group Total Controls (n=360)
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Age (year)
63.4±9.0
62.6±8.7
0.210
Gender (male/female) BMI (kg/m2 )
239/157 24.3±2.15
211/149 24.0±2.08
0.626 0.057
Hypertension (%) Diabet esmellits (%)
234 (59.1) 106 (26.8)
112 (31.1) 60 (16.7)
<0.001 <0.001
Smoking (%)
103 (26.0)
41 (11.4)
<0.001
Alcohol drinker (%) SBP (mmHg)
97 (24.5) 143±18
37 (10.3) 132±15
<0.001 <0.001
DBP (mmHg) Blood glucose (mmol/l)
84±10 6.42±2.17
79±9 5.72±1.14
<0.001 <0.001
Total cholesterol (mg/dl) Triglycerides (mmol/l)
4.76±1.05 1.59±1.03
4.44±0.87 1.32±0.65
<0.001 <0.001
HDL (mmol/l) LDL (mmol/l)
1.14±0.31 3.19±0.97
1.15±0.27 2.83±0.76
0.494 <0.001
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Cases (n=396)
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Data are presented as the mean±SD or percent frequency. n: Number; SD: standard deviation; BMI, body-mass index; SBP, systolic blood pressure; DBP, diastolic blood pressure; HDL-C,
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high-density lipoprotein cholesterol; LDL-C, low-density lipoprotein cholesterol. P values from Student's t-test or χ2. Table 2 Genotype and allele distributions for the patients with ischemic stroke and the control group Total
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Cases
n (%)
rs12218
Genotype
Allele rs2468844 Genotype Allele
Controls n (%)
Single analysis P
OR (95%CI)
211(53.3) 150(37.9)
223(61.9) 119(33.1)
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35(8.8) 572(72.2)
18(5.0) 565(78.5)
0.018
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220(27.8)
155(21.5)
0.005
AA
338(85.4)
328(91.1)
AG
58(14.6)
32(8.9)
A
734(92.7)
688(95.6)
0.066
regression analysis P OR (95%CI) 0.045
0.023
TT CT
Multiple logistic
Reference 1.33(0.98-1.81)
Reference 0.126
1.25(0.91-1.79)
2.06(1.13-3.74) Reference
0.044
1.64(1.03-3.29)
1.40(1.11-1.78)
0.048
Reference
Reference 0.016
1.76(1.11-2.78) Reference
1.27(1.01-1.68)
Reference 0.241
1.37(0.81-2.31)
Reference
1.34(0.81-2.22) G 58(7.3) 32(4.4) 0.019 1.70(1.09-2.65) 0.259 Data are adjusted for hypertension, diabetes mellitus, smoking and alcohol consumption, systolic
ACCEPTED MANUSCRIPT blood pressure (SBP), diastolic blood pressure (DBP), blood glucose, total plasma cholesterol, triglycerides, and plasma LDL; OR, odds ratio; CI, credibility interval.
Table 3 Genotype and allele distributions for the patients with the male and the control group males Case n (%)
rs2468844 Genotype
P
OR (95%CI)
T C
340(71.1) 138(28.9)
332(78.7) 90(21.3)
AA AG
202(84.5) 37(15.5)
192(91.0) 19(9.0)
PT
131(62.1) 70(33.2) 10(4.7)
0.141 0.020
Reference 1.35(0.91-2.00) 2.52(1.16-5.47)
0.010
Reference 1.50(1.1-2.03)
0.040
Reference 1.85(1.03-3.33)
1.47(0.91-2.37)
0.036
2.66(1.07-6.62)
0.010
Reference 1.57(1.09-2.26)
Reference 0.156
1.62(0.83-3.15)
A 441(92.3) 403(95.5) Reference Reference 1.56(0.82-2.96) G 37(7.7) 19(4.5) 0.047 1.78(1.01-3.14) 0.170 Data are adjusted for hypertension, diabetes mellitus, smoking and alcohol consumption, systolic
MA
Allele
Reference
0.115
RI
125(52.3) 90(37.7) 24(10.0)
regression analysis P OR (95%CI) 0.049
0.039 TT CT CC
Multiple logistic
SC
Allele
n (%)
Single analysis
NU
rs12218 Genotype
Control
blood pressure (SBP), diastolic blood pressure (DBP), blood glucose, total plasma cholesterol,
ED
triglycerides, and plasma LDL; OR, odds ratio; CI, credibility interval.
Table 4 Genotype and allele distributions for the patients with the female and the control group females
n (%) rs12218
Allele
n (%)
TT CT
86(54.8) 60(38.2)
92(61.7) 49(32.9)
CC T C
11(7) 232(73.9) 82(26.1)
8(5.4) 233(78.2) 65(21.8)
AA
136(86.6)
136(91.3)
AC C
Genotype
Control
EP T
Case
Single analysis P
OR (95%CI)
0.770
0.455 0.269 0.429 0.213
Multiple logistic regression analysis P OR (95%CI)
Reference 1.31(0.81-2.11) 1.47(0.56-3.83) Reference 1.27(0.87-1.84)
Reference 0.999
1.00(0.57-1.77)
0.477
0.64(0.19-2.18)
Reference 0.660
0.9(0.58-1.42)
rs2468844 Genotype
AG 21(13.4) 13(8.7) 0.199 Allele A 293(93.3) 285(95.6) G 21(6.7) 13(4.4) 0.213 Data are adjusted for hypertension, diabetes mellitus,
Reference
Reference
1.04(0.42-2.55) 1.62(0.78-3.36) 0.933 Reference Reference 0.940 1.04(0.44-2.47) 1.57(0.77-3.2) smoking and alcohol consumption, systolic
blood pressure (SBP), diastolic blood pressure (DBP), blood glucose, total plasma cholesterol, triglycerides, and plasma LDL; OR, odds ratio; CI, credibility interval.
ACCEPTED MANUSCRIPT Table 5 Genotype and allele distributions for the patients with the LAA and the control group LAA
Allele
Control
n (%)
n (%)
Single analysis P
OR (95%CI)
regression analysis OR (95%CI)
TT
85(48)
223(61.9)
CT
70(39.5)
119(33.1)
0.028
Reference 1.54(1.05-2.27)
0.248
1.32(0.82-2.11)
CC
22(12.4)
18(5)
0.001
3.21(1.64-6.27)
0.044
2.34(1.02-5.33)
T
240(67.8)
565(78.5)
C
114(32.2)
155(21.5)
AA AG
148(83.6) 29(16.4)
328(91.1) 32(8.9)
A
325(91.8)
688(95.6)
Reference
Reference
0.000
1.73(1.30-2.30)
0.011
Reference 2.01(1.17-3.44)
RI
0.036
Reference 1.45(1.02-2.06)
Reference
0.195
SC
Allele
P
0.046
0.001
rs2468844 Genotype
Multiple logistic
PT
rs12218 Genotype
Case
Reference
1.54(0.8-2.96)
Reference
NU
1.49(0.79-2.78) G 29(8.2) 32(4.4) 0.014 1.92(1.14-3.23) 0.214 Data are adjusted for hypertension, diabetes mellitus, smoking and alcohol consumption, systolic
blood pressure (SBP), diastolic blood pressure (DBP), blood glucose, total plasma cholesterol,
MA
triglycerides, and plasma LDL; OR, odds ratio; CI, credibility interval.
Table 6 Genotype and allele distributions for the patients with the SAO and the control group SAO
rs12218 TT
126(57.5)
223(61.9)
CT CC
80(36.5) 13(5.9)
119(33.1) 18(5)
T C
332(75.8) 106(24.2)
565(78.5) 155(21.5)
rs2468844 Genotype
AA
190(86.8)
328(91.1)
Allele
AG A
29(13.2) 409(93.4)
32(8.9) 688(95.6)
Allele
AC C
Genotype
n (%)
EP T
n (%)
Control
ED
Case
Single analysis P
OR (95%CI)
Multiple logistic regression analysis P 0.739
0.566 Reference 0.341 0.519
1.19(0.83-1.7) 1.28(0.61-2.7)
0.291
Reference 1.16(0.88-1.54)
Reference 0.478
1.16(0.77-1.75)
0.660
1.21(0.51-2.87)
Reference 0.448
Reference 0.100
OR (95%CI)
1.56(0.92-2.67) Reference
1.13(0.82-1.57)
Reference 0.691
1.13(0.61-2.11)
Reference
1.12(0.62-2.05) G 29(6.6) 32(4.4) 0.110 1.52(0.91-2.56) 0.702 Data are adjusted for hypertension, diabetes mellitus, smoking and alcohol consumption, systolic
blood pressure (SBP), diastolic blood pressure (DBP), blood glucose, total plasma cholesterol, triglycerides, and plasma LDL; OR, odds ratio; CI, credibility interval.
ACCEPTED MANUSCRIPT Highlights
PT RI SC NU MA ED EP T
Our study focused on Northern Chinese Han population. The SAA gene polymorphisms were associated with susceptibility to ischemic stroke. The SNPs of SAA gene had a significant in males. The SNPs of SAA gene had a significant in atherosclerosis ischemic stroke subtype.
AC C