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PHD : Parasites, Hosts and Diseases

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Original Article

Subsets of Inflammatory Cytokine Gene Polymorphisms are Associated with Risk of Carcinogenic Liver Fluke Opisthorchis viverrini-Associated Advanced Periductal Fibrosis and Cholangiocarcinoma

The Korean Journal of Parasitology 2017;55(3):295-304.
Published online: June 30, 2017

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*Corresponding author (banchob@kku.ac.th)
• Received: April 18, 2017   • Revised: May 8, 2017   • Accepted: May 11, 2017

Copyright © 2017 by The Korean Society for Parasitology and Tropical Medicine

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Subsets of Inflammatory Cytokine Gene Polymorphisms are Associated with Risk of Carcinogenic Liver Fluke Opisthorchis viverrini-Associated Advanced Periductal Fibrosis and Cholangiocarcinoma
Korean J Parasitol. 2017;55(3):295-304.   Published online June 30, 2017
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Subsets of Inflammatory Cytokine Gene Polymorphisms are Associated with Risk of Carcinogenic Liver Fluke Opisthorchis viverrini-Associated Advanced Periductal Fibrosis and Cholangiocarcinoma
Korean J Parasitol. 2017;55(3):295-304.   Published online June 30, 2017
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Subsets of Inflammatory Cytokine Gene Polymorphisms are Associated with Risk of Carcinogenic Liver Fluke Opisthorchis viverrini-Associated Advanced Periductal Fibrosis and Cholangiocarcinoma
Image Image Image
Fig. 1 Representative amplicons and digestion fragments corresponding to gene polymorphisms. (A) 190 and 114 bp fragments specific for C allele, 304 bp fragment specific for T allele of IL-1b-511C/T polymorphism. (B) 233 bp and 54 bp bands specific for G allele, 122 bp, 111 bp, and 54 bp bands specific for C allele of IL-6-174G/C polymorphism. C) 262 bp fragment specific for T and A alleles of IFN-γ +874T/A polymorphism. (D) 427 bp fragment specific for A allele, 231 and 196 bp fragments specific for G allele of LT-α +252A/G polymorphism. (E) 107 bp fragment specific for A allele, 87 bp fragments specific for G allele of TNF-α −308G/A polymorphism. M, 50 bp DNA marker.
Fig. 2 Net cytokine profile of OvES stimulated PBMCs in APF−, APF+, and CCA patients. The Wilcoxon Matched-paired Signed rank test was used to assess the statistical significance; *P<0.05.
Fig. 3 The net production of IL-1β, IL-6, IFN-γ, LT-α, and TNF-α, and their genotype polymorphisms in APF− (n=200), APF+ (n=200), and CCA patients (n=110). wt, wild type; hetero, heterozygous; var, variant.
Subsets of Inflammatory Cytokine Gene Polymorphisms are Associated with Risk of Carcinogenic Liver Fluke Opisthorchis viverrini-Associated Advanced Periductal Fibrosis and Cholangiocarcinoma

Primer sequences, restriction enzymes, and size of digestion products in the PCR-RFLP and AS-PCR analyses

Gene/SNP rsn wt/var Region Oligonucleotide primers RE Digestion products (bp)
IL-1β/−511 rs16944 C/T Promoter F (5′-TGG CAT TGA TCT GGT TCA TC-3′)
R (5′-GTT TAG GAA TCT TCC CAC TT-3′)
Ava I wt: 190, 114
var: 304
IL-6/−174 rs1800795 G/C Promoter F (5′-GGG CTG CGA TGG AGT CGA AG-3′)
R (5′-TCC CTC ACA CAG GGC TCG-3′)
Nla III wt: 233, 54
var: 122. 111, 54
IFN-γ/+874 rs2430561 T/A Intron 1 Common primer: 5′TCAACAAAGCTGATACTCCA-3′, T allele primer: 5′TTCTTACAACACAAAATCAAATCT-3′, A allele primer: 5′TCTTACAACACAAAATCAAATCA-3′
LT-α/+252 rs909253 A/G intron F (5′-CCG TGC TTC GTG CTT TGG ACT A-3′)
R (5′-AGA CGT TCA GGT GGT GTA AT-3′)
Nco I wt: 427
var: 231, 196
TNF-α/−308 rs1800629 G/A promoter F (5′-TCC TCC CTG CTC CGA TTA CG-3′)
R (5′-AGG CAA TAG GTT TTG AGG GCC-3′)
Nco I wt: 87
var: 107

SNP, Single nucleotide polymorphism; rsn, reference SNP number; wt, wild type; var, variant; RE, restriction enzyme; bp, base pair

Clinicopathological characteristics of cholangiocarcinoma (CCA) patients and non-CCA controls

Demographics APF− (n=200) APF+ (n=200) CCA (n=110)
Sex
 Male (%) 91 (45.5) 92 (46.0) 72 (65.5)
 Female (%) 109 (54.5) 108 (54.0) 38 (34.5)

Age (years)
 Mean±SD 46.8 ±7.7 47.4±7.5 55.9±10.3
 Range 20–60 22–60 40–76

Age (in year; %)
 ≤ 30 6 (3.0) 5 (2.5) 1 (0.9)
 31–40 34 (17.0) 33 (16.5) 5 (4.6)
 41–50 86 (43.0) 83 (41.5) 20 (18.2)
 ≥ 51 74 (37.0) 79 (39.5) 84 (46.4)

Cytokine levels as established in PBMCs pulsed with excretory secretory profcuts of the liver fluke Opisthorchis viverrini and the net cytokine production and genotype polymorphisms

Cytokine levels, pg/ml
Wild type Heterozygous Variant
IL-1β 268.4±47.7 315.4±35.9 303.1±41.1
IL-6 3,973.1±763.2 4,221.6±434.8 4,323.7±440.5
IFN-γ 514.9±140.3 397.5±117.4 239.9±65.2
LT-α 67.3±17.9 187.4±36.0* 153.7±36.7*
TNF-α 362.6±68.8 498.8±127.1 466.7±67.9

Cytokine levels presented as mean±SD;

*P<0.05; n=510.

Comparison of genotypes and allele frequencies for IL-1β, IL-6, IFN-γ, LT-α, and TNF-α in CCA cases and the participants in the APF+ and the APF− groups

Cytokine/Genotype/Allele APF−N(%) APF+N(%) CCA N(%) APF− vs. APF+ APF− vs. CCA APF+ vs. CCA

OR (95% CI) OR (95% CI) OR (95% CI)
IL-1β (−511C/T)
 CC 38 (19.0) 36 (18.0) 18 (16.1) 1 1 1
 CT 100 (50.0) 93 (46.5) 48 (43.6) 0.98 (0.50–1.74) 1.01 (0.50–2.09) 1.03 (0.50–2.14)
 TT 62 (31.0) 71 (35.5) 44 (40.0) 1.20 (0.65–2.22) 1.49 (0.72–3.16) 1.23 (0.59–2.61)
 C 176 (44.0) 165 (41.2) 84 (38.2) 1 1 1
 T 244 (56.0) 235 (58.8) 136 (61.8) 1.02 (0.77–1.36) 1.16 (0.82–1.65) 1.13 (0.80–1.61)

IL-6 (−174G/C)
 GG 112 (56.0) 124 (62.0) 32 (29.1) 1 1 1
 GC 67 (33.5) 59 (29.5) 45 (40.9) 0.79 (0.50–1.25) 2.35 (1.31–4.21)* 2.95 (1.64–5.31)*
 CC 21 (10.5) 17 (8.5) 33 (30.0) 0.73 (0.34–1.53) 5.50 (2.65–11.41)* 7.52 (3.52–16.19)*
 G 291 (72.7) 307 (76.7) 109 (49.5) 1 1 1
 C 109 (27.3) 93 (23.3) 111 (50.5) 0.81(0.58–1.13) 0.26 (0.12–0.52) 3.36 (2.32–4.85)*

IFN-γ (+874T/A)
 TT 61 (30.5) 26 (13.0) 23 (20.9) 1 1 1
 TA 74 (37.0) 98 (49.0) 33 (30.0) 3.10 (1.73–5.61)* 1.18 (0.60–2.34) 0.38 (0.18–0.80)
 AA 65 (32.5) 76 (38.0) 54 (49.1) 2.74 (1.50–5.04)* 2.20 (1.13–4.20)* 0.80 (0.39–1.64)
 T 196 (49.0) 150 (37.5) 79 (35.9) 1 1 1
 A 204 (51.0) 250 (62.5) 141 (64.1) 1.60 (1.19–2.14)* 1.71 (1.20–2.44)* 1.07 (0.75–1.5)

LT-α (+252A/G)
 AA 60 (30.0) 47 (23.5) 24 (21.8) 1 1 1
 AG 74 (37.0) 93 (46.5) 54 (49.1) 1.60 (0.95–2.65) 1.84 (0.97–3.45) 1.17 (0.60–2.16)
 GG 66 (33.0) 60 (30.0) 32 (29.1) 1.16 (0.66–2.01) 1.21 (0.61–2.40) 1.04 (0.51–2.11)
 A 194 (48.5) 187 (46.8) 102 (46.4) 1 1 1
 G 206 (51.5) 213 (53.2) 118 (53.6) 1.07 (0.80–1.43) 1.08 (0.77–1.53) 1.01 (0.72–1.43)

TNF-α (−308G/A)
 GG 34 (17.0) 35 (17.5) 17 (15.5) 1 1 1
 GA 54 (27.0) 46 (23.0) 13 (11.8) 0.82 (0.42–1.60) 0.48 (0.18–1.20) 0.58 (0.22–1.47)
 AA 112 (56.0) 119 (59.5) 80 (72.7) 1.03 (0.58–1.83) 1.42 (0.71–2.92) 1.38 (0.69–2.82)
 G 122 (30.5) 116 (29.0) 47 (21.4) 1 1 1
 A 278 (69.5) 284 (71.0) 173 (78.6) 1.07 (0.78–1.47) 1.61 (1.08–2.43)* 1.50 (1.00–2.26)*

*Significant at P-value<0.05.

Table 1 Primer sequences, restriction enzymes, and size of digestion products in the PCR-RFLP and AS-PCR analyses

SNP, Single nucleotide polymorphism; rsn, reference SNP number; wt, wild type; var, variant; RE, restriction enzyme; bp, base pair

Table 2 Clinicopathological characteristics of cholangiocarcinoma (CCA) patients and non-CCA controls
Table 3 Cytokine levels as established in PBMCs pulsed with excretory secretory profcuts of the liver fluke Opisthorchis viverrini and the net cytokine production and genotype polymorphisms

Cytokine levels presented as mean±SD;

P<0.05; n=510.

Table 4 Comparison of genotypes and allele frequencies for IL-1β, IL-6, IFN-γ, LT-α, and TNF-α in CCA cases and the participants in the APF+ and the APF− groups

Significant at P-value<0.05.