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Choi, Lee, Kim, and Ki: Association between Polymorphisms in Toll-like Receptor 9 Gene and Outcomes after Ischemic Stroke
Original Article

J Bacteriol Virol 2015;45(3):242-249.

Published online: 9 February 2015

DOI: https://doi.org/10.4167/jbv.2015.45.3.242

Association between Polymorphisms in Toll-like Receptor 9 Gene and Outcomes after Ischemic Stroke

Jay Chol Choi1, Keun-Hwa Lee2, ∗, Young Ree Kim3, Chang-Seok Ki4

1Department of Neurology, Jeju National University, Jeju, Korea

2Department of Microbiology and Immunology, Jeju National University, Jeju, Korea

3Department of Laboratory Medicine, Jeju National University, Jeju, Korea

4Department of Laboratory Medicine and Genetics, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea

∗Corresponding author: Keun Hwa Lee Ph.D. Department of Microbiology and Immunology, Jeju National University School of Medicine Jeju, 690-756, Korea. Phone: +82-64-754-8111, Fax: +82-64-705-2687, e-mail: yomust7@jejunu.ac.kr

∗∗ This research was supported by the research grant from Jeju National University Hospital.

The biospecimens and data used in this study were provided by the Biobank of Jeju University Hospital, a member of Korea Biobank Network (No. A-01).

Received 19 June 201527 June 2015       Accepted 29 June 2015

Copyright © 2015 Journal of Bacteriology and Virology

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Several evidences suggested that Toll-like receptor 9 (TLR9) plays an important role in atherosclerosis and neuroprotection but the association between the TLR9 and risk for stroke or outcomes after stroke has not been investigated. The aim of the present study was to investigate the association between TLR9 polymorphisms and the risk for ischemic stroke using a case-control study design. We also explored the correlation between the polymorphisms and outcomes after stroke. We enrolled consecutive Korean stroke patients and controls without history of stroke. Four polymorphisms, namely c.-1486T>C, c.-1237C>T, c.1174A>G, and c.2848G>A were examined using polymerase chain reaction followed by direct sequencing. Initially we examined 193 stroke patients and the same number of healthy adults who had no history of stroke as controls. Due to deviation from Hardy-Weinberg equilibrium of initial controls, we performed genetic analysis of two polymorphisms (c.1174A>G and c.2848G>A) for additional 165 controls. The genotype frequency of four polymorphisms did not differ significantly between stroke patients and controls in unadjusted analysis. The variant allele (C) in c.-1486 locus was associated with significantly increased chance of favorable functional outcome at three month after stroke (OR 2.32, 95% CI 1.02~5.26, p = 0.043).

Keywords: Toll-like receptor, Polymorphism, Ischemic stroke, Outcomes

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Table 1.
The sequences of the primers used for the genotypes of four polymorphisms in this study
Locus Direction Sequence
c.-1486T>C Forward 5′-TCATTCAGCCTTCACTCAGAAA-3′
  Reverse 5′-ACCTCCCACCCCAGATCT-3′
c.-1237C>T Forward 5′-TAAGAAGGCTGGATGGCCCT-3′
  Reverse 5′-GGGACCTGCCACCCG-3′
c.1174A>G Forward 5′-AATTCTGAGTCCAAGACTGGGTCT-3′
  Reverse 5′-GCTGGAGCTCACAGGGTAGG-3′
c.2848C>T Forward 5′-AGGCTGAGGTGGCGCA-3′
  Reverse 5′-CGGAGATGTTTGCCCAGCT-3′
Table 2.
Baseline characteristics of 193 stroke patients and 193 controls
  Control (n=193) Stroke (n=193) p
Demographic characteristics      
  Age, years 67±12 65±12 <0.001
  Sex, men 116 (60.1) 116 (60.1) 1.000
  BMI, kg/m2 24.8±3.2 24.1±3.3 0.068
Vascular risk factors      
  Hypertension 67 (34.7) 120 (62.2) <0.001
  Diabetes mellitus 30 (15.5) 57 (29.5) 0.001
  Smoking 58 (30.1) 84 (43.5) 0.006
Laboratory findings      
  Hemoglobin 13.1±2.1 13.7±2.1 0.009
  Fasting blood glucose, mg/dl 110±46 131±53 <0.001
  Systolic blood pressure, mmHg 131±17 156±26 <0.001
  Diastolic blood pressure, mmHg 79±12 88±14 <0.001

BMI, body mass index. Data are mean ± SD values or n (%). Analyzed by t-test, Wilcoxon signed rank test, or chi-square test according to the type of variables

Table 3.
Genotype distribution of the c.-1486T>C and c.-1237T>C promotor polymorphisms between stroke patients and controls and the unadjusted and adjusted risk of stroke by the genotypes
Locus Genotype Control (%) (n=193) Stroke (%) (n=193) Odd ratio (95% CI) p Adjusted OR∗ (95% CI) p
c.-1486T>C TT 62 (32.1) 76 (39.4) Reference - Reference -
  TC 107 (55.4) 87 (45.1) 0.66 (0.42~1.05) 0.066 0.85 (0.46~1.55) 0.594
  CC 24 (12.4) 30 (15.5) 1.02 (0.52~2.02) 0.952 1.28 (0.85~1.92) 0.242
  TC+CC 131 (67.9) 117 (60.6) 0.73 (0.47~1.13) 0.137 1.01 (0.58~1.76) 0.966
  Minor allele frequency 77 (40.0) 73 (38.0) 0.92 (0.60~1.41) 0.676    
c.-1237T>C TT 187 (96.9) 185 (95.9) Reference - Reference -
  TC 6 (3.1) 8 (4.1) 1.35 (0.40~4.81) 0.586 2.19 (0.53~9.09) 0.279
  CC 0 0 NA NA NA NA
  TC+CC 6 8 1.35 (0.40~4.81) 0.586 2.19 (0.53~9.09) 0.279
  Minor allele frequency 4 (2.0) 4 (2.0) 1.00 (0.18~5.45) 1.000    

∗ Adjusted for age, sex, BMI, history of hypertension, diabetes mellitus, smoking, hemoglobin, and fasting blood glucose

Table 4.
Genotype distribution of the c.1174A>G and c.2848C>T polymorphisms between stroke patients and controls and risk of stroke by the genotype
Locus Genotype Control (n=358) Stroke (n=193) Odd ratio (95% CI) p Adjusted OR∗ (95% CI) p
c.1174A>G AA 118 (33.0) 71 (36.8) Reference - Reference -
  AG 187 (52.2) 89 (46.1) 0.79 (0.53~1.19) 0.236 1.00 (0.60~1.67) 0.999
  GG 53 (14.8) 33 (17.1) 1.03 (0.59~1.80) 0.898 1.18 (0.84~1.66) 0.339
  AG+GG 240 (67.0) 122 (63.2) 0.84 (0.58~1.24) 0.367 1.08 (0.67~1.73) 0.761
  Minor allele frequency 147 (40.0) 77 (40.0) 0.95 (0.66~1.38) 0.790    
c.2848C>T CC 115 (32.1) 75 (38.9) Reference - Reference -
  CT 186 (52.0) 86 (44.6) 0.71 (0.47~1.06) 0.081 1.04 (0.62~1.73) 0.881
  TT 57 (15.9) 32 (16.6) 0.86 (0.49~1.49) 0.573 1.13 (0.81~1.57) 0.474
  CT+TT 243 (67.9) 118 (61.1) 0.74 (0.51~1.09) 0.113 1.09 (0.68~1.74) 0.730
  Minor allele frequency 150 (42.0) 75 (39.0) 0.74 (0.51~1.09) 0.113    

∗ Adjusted for age, sex, BMI, history of hypertension, diabetes mellitus, smoking, hemoglobin, and fasting blood glucose

Table 5.
Baseline NIHSS score by TLR polymorphisms
  Unadjusted analysis Adjusted analysis∗
Polymorphism Wild Variant p Wild Variant p
c.-1486T>C 5.1 (3.7~6.5) 4.4 (3.5~5.3) 0.283 4.3 (2.0~6.5) 3.4 (1.3~5.6) 0.269
c.-1237T>C 4.6 (3.8~5.4) 6.4 (1.8~11.0) 0.236 3.9 (1.8~6.0) 5.5 (0.7~10.2) 0.441
c.1174A>G 4.8 (3.9~5.6) 4.2 (2.5~6.0) 0.390 4.0 (1.9~6.1) 2.9 (0.3~5.5) 0.281
c.2848C>T 5.1 (3.7~6.5) 4.4 (3.5~5.3) 0.634 4.1 (1.8~6.3) 3.5 (1.3~5.71) 0.498

Values are mean (95% CI) or adjusted mean (95% CI)

∗ Adjusted for age, sex, onset to arrival time, hypertension, diabetes mellitus, hyperlipidemia, atrial fibrillation, smoking, fasting blood glucose and TOAST classification. NIHSS, National Institutes of Health Stroke Scale; mRS, modified Rankin score.

Table 6.
Unadjusted and adjusted odd ratios for favorable clinical outcomes at three month by TLR genotypes
Polymorphism Unadjusted OR (95% CI) p Adjusted OR (95% CI) p
c.-1486T>C 1.60 (0.81~3.15) 0.142 2.32 (1.02~5.26) 0.043
c.-1237T>C 0.47 (0.06~3.64) 0.355 1.24 (0.11~13.38) 0.861
c.1174A>G 1.23 (0.48~3.45) 0.653 1.11 (0.36~3.38) 0.859
c.2848C>T 1.17 (0.59~2.31) 0.620 1.74 (0.77~3.90) 0.180

Adjusted for age, sex, onset to arrival time, hypertension, diabetes mellitus, hyperlipidemia, smoking, atrial fibrillation, fasting blood glucose, TOAST classification, and initial NIHSS scores.

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