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  • Research article
  • Open Access
  • Open Peer Review

Toll-like receptor 4 Asp299Gly and Thr399Ile polymorphisms: New data and a meta-analysis

  • 1Email author,
  • 1,
  • 1,
  • 3,
  • 2 and
  • 1
BMC Gastroenterology201414:206

https://doi.org/10.1186/s12876-014-0206-x

  • Received: 22 June 2014
  • Accepted: 24 November 2014
  • Published:
Open Peer Review reports

Abstract

Background

The pathogenesis of inflammatory bowel disease (IBD) involves interactions between the host genetic susceptibility, intestinal microflora and mucosal immune responses through the pattern recognition receptor. Polymorphisms in toll-like receptor 4 (TLR4) induce an aberrant immune response to indigenous intestinal flora, which might favor IBD development. In this study, we aimed to determine whether TLR4 gene was associated with Crohn’s disease (CD) and ulcerative colitis (UC) among Moroccan patients, and evaluated its correlation with clinical manifestation of the disease.

Methods

The study population comprised 117 patients with IBD and 112 healthy unrelated blood donors. TLR4 polymorphisms: Asp299Gly and Thr399Ile were genotyped by polymerase chain reaction-restriction fragment length polymorphism. PCR products were cleaved with Nco I for the Asp299Gly polymorphism and Hinf I for the Thr399Ile polymorphism. Meta-analysis was performed to test the association of 299Gly and 399Ileu carriage with CD, UC and the overall IBD risk.

Results

Our study revealed that the frequency of Asp299Gly and Thr399Ile did not differ significantly between patients and controls in the Moroccan population. However, meta-analysis demonstrated significantly higher frequencies of both Asp299Gly and Thr399Ile SNPs in IBD and CD and for 399Ileu carriage in UC patients.

Conclusion

The meta-analysis provides evidence that TLR4 polymorphisms confer a significant increased risk for the overall IBD development.

Keywords

  • Toll-like receptor 4
  • Inflammatory bowel disease
  • Moroccan patients

Background

Inflammatory bowel disease (IBD) is an idiopathic and chronic multifactorial disease of the gastrointestinal tract. Although the precise etiology of IBD is unclear, several factors that play a crucial role in disease pathogenesis such as commensal bacterial flora and genes related to the host immune response have been identified [1],[2].

Toll-like receptors are pattern recognition receptors through which host recognizes microbial conserved molecular motifs that are broadly shared by pathogens, therefore they are very important for the regulation of mucosal innate immune responses to intestinal microbes. Perturbations in individual TLR biological signaling can prime to a number of different outcomes and elucidate a system of regulation within the intestine in which each TLR plays a largely non-redundant role in mucosal immunity.

TLR4 gene, the first mammalian TLR identified is located on the long arm of human chromosome 9q32-33 [3]. It encodes the transmembrane receptor that initiates the innate immune response to common gram-negative bacteria [4],[5].

TLR4 is the major transducer of lipopolysaccharide (LPS) and binds specifically lipid A moiety. Signal transduction through TLR4 in combination with CD14, and MD-2 leads to activation of the nuclear factor-κB (NF-κB) system through the MyD88-dependent and MyD88-independent pathways and subsequent expression of inflammatory genes encoding cytokines and cell conjugation molecules as part of host defense mechanisms [6]-[9].

Under healthy conditions TLR4 is only minimally expressed in lamina propria mononuclear cells (LPMNCs) and intestinal epithelial cells which are partly tolerant to LPS, thus preventing an exaggerated immune response mediated by the large number of bacteria in the intestinal lumen and maintaining a basal state of activation [10],[11].

However, TLR4 expression is upregulated in human IBD colitis, maximizing responsiveness to the environment and reflecting an aberrant state of activation [12]-[15]. Higher levels of TLR4 mRNA and protein were found in the inflamed colonic mucosa in pediatric IBD patients [16]. Two common co-segregating polymorphisms affecting the extracellular domain of the TLR4 (Asp299Gly and Thr399Ile) have been described in humans. Individuals heterozygous for these mutations have a blunted response to inhaled LPS [17]. These polymorphisms are thought to be associated with increased susceptibility to IBD. However, population studies reveal discordant results in geographical distribution.

Thereby, we sought to investigate whether Asp299Gly and Thr399Ile single nucleotide polymorphisms of the gene encoding the TLR4 determine susceptibility to IBD in Moroccan patients and assessed their influence on phenotype expression.

Methods

Study population

Enrolled in this study were 117 IBD Moroccan patients (83 CD; 34 UC) and 112 healthy unrelated blood donors. The diagnosis of CD or UC was established according to conventional clinical, endoscopic, radiological and histological criteria as previously reported [18],[19]. CD was classified according to the Montreal classification [20].

The case report form included questions on disease phenotype and location, age at diagnosis, toxic behavior and other clinical features. The ethics committee of the Faculty of Medicine and Pharmacy of Casablanca approved the study and a written informed consent was obtained from all human subjects.

Molecular analysis of TLR4polymorphisms

Genomic DNA was extracted from peripheral blood leukocytes using the salting out procedure. Two single nucleotide variations, corresponding to two amino acid polymorphisms for TLR4, were analyzed: the Asp299Gly (896A/G), rs4986790 and the Thr399Ile (1196C/T), rs4986791.

Typing of the polymorphisms was performed using polymerase chain reaction (PCR) restriction fragment length polymorphism analysis (RFLP).

Upstream and downstream primers used for the PCR amplification were:

F: (5′- AGCATACTTAGACTACTACCTCCATG-3′),

R: (5′- GAGAGATTTGAGTTTCAATGTGGG-3′) for TLR4Asp299Gly

And F: (5′-GGTTGCTGTTCTCAAAGTGATTTTGGGAGAA-3′),

R:(5′-GGAAATCCAGATGTTCTAGTTGTTCTAAGCC-3′) for TLR4Thr399Ile.

Reactions were performed in a 25 μl volume containing 200 μM of each dNTP (0.5 μl of dNTP mix, 10 mM each), 0.2 μM of each of the forward and reverse primers (0.5 μl of each 10 μM primers), 2 mM MgCl2 (1 μl of MgCl2, 50 mM) and 1 U of Taq DNA polymerase (1 μl of 1U/μl enzyme), 1× PCR buffer (2.5 μl of 10× PCR buffer).

PCR conditions comprised 5 min at 95°C then 35 cycles of denaturing were performed at 95°C for 30 s, annealing at 55°C (Asp299Gly) and at 53°C (Thr399Ile) for 1 min, 72°C for 30 s. A final extension phase of 72°C for 10 minutes was used.

PCR products were cleaved overnight at 37°C with Nco I for Asp299Gly polymorphism and HinfI for Thr399Ile polymorphism (Biolabs). The digests were run on a 3% agarose gel and visualized under UV light using ethidium bromide.

The mutant alleles (GG)/(TT) contained an Nco I/ Hinf I restriction site for the Asp299Gly/Thr399Ile polymorphisms respectively, allowing RFLP analysis of the digested products. Digestion at the Nco I site yields fragments of 168 and 20 bp, the one at Hinf I site yields fragments of 98 and 26 bp. The wild-type allele for both polymorphisms remained uncut.

Statistical analysis

The data were analyzed with MedCalc 11.6. Chi-square test was used to compare the allele and genotype frequencies between disease and control groups. The Fisher’s exact test was used when appropriate. The observed genotype frequencies were compared with the predicted frequencies by the Hardy–Weinberg equilibrium.

The average age was determined by the rank sum test. Associations between genotypes and risk of IBD were estimated by calculating odds ratio (OR) with confidence interval of 95% (CI). P values less than 0.05 were considered significant in disease risk association tests. The χ2 test or Fisher test was used to correlate the TLR4 polymorphisms and clinical parameters. The Bonferroni correction method was applied for correction for multiple testing in sub-phenotype analysis; The phenotype genotype correlation was considered statistically significant if the p value was less than 0.005 for CD and 0.007 for UC. According to Power Calculator for Genetic Studies 2006 software (http://www.sph.umich.edu/csg/abecasis/CaTS/), this study had 15% of power to detect an OR of 1.5.

TLR4meta-analysis

Inclusion and exclusion criteria

Genetic association studies were included in our meta-analysis if they met the following criteria:
  1. (a)

    Studies that evaluated the association between the TLR4 Asp299Gly, Thr399Ile polymorphisms and IBD, (b) A case–control study design, (c) The study reported sufficient data to calculate allele frequencies, odds ratios and confidence intervals of cases and controls for carriage of the TLR4 299Gly and 399Ile alleles.

     

While major exclusion criteria were: (a) case-only study and review articles (b) absence of the mutant allele in both cases and controls, (c) studies without the raw data of the TLR4 Asp299Gly and Thr399Ile genotypes.

Pooled studies for case–control meta-analysis

Twenty-four case–control studies were identified through the literature search.

Asp299Gly polymorphism: According to the inclusion criteria, twenty studies were retrieved in CD meta-analysis (Table 1), four of them contained more than one cohort [21]-[24]. UC meta-analysis reported data from 13 of the included studies (Table 2); two of them contained more than one cohort [21],[22]. Six studies met one of the exclusion criteria [25]-[30].

Thr399Ile Polymorphism: twelve studies comprising 2466 cases and 2210 controls were included in CD meta-analysis (Table 3), and nine of them in UC meta-analysis with 1358 cases and 1773 controls (Table 4).

A meta-analysis combining CD and UC patients for the two tested SNPs: Asp299Gly and Thr399Ile included 13 and 9 studies respectively (Tables 5 and 6).

The risk of IBD associated with the TLR4 polymorphism was estimated for each study by odds ratio (OR) and 95% confidence interval (95% CI).

The meta-ORs were estimated using a fixed-effects model with the wild-type allele as reference group. Genetic heterogeneity was tested by Cochran’s (Q) test, I2 statistics was used to quantify the between-study heterogeneity effect. When a significant Q test (Q > 0.10; I2 > 50%) indicated heterogeneity across studies, data were recombined using a random-effects model to estimate common ORs. The meta-analyses were conducted by Review Manager 5.0 and MedCalc bvba 12.3.0 softwares.
Table 1

Pooled analysis of studies exploring the role of TLR4 Asp299Gly in CD

Study

Cases (Events/Total)

Controls (Events/Total)

Odds ratio

95% CI

P value

Arnott et al. 2004 [ [31]]

50/468

33/378

1,25

0,79 to 1,98

 

Franchimont et al. 2004 [ [32]] (1)

73/668

14/278

2,31

1,28 to 4,17

 

Franchimont et al. 2004 [ [32]] (2)

26/226

14/278

2,45

1,25 to 4,81

 

Torok et al. 2004 [ [33]]

14/204

12/290

1,70

0,77 to 3,77

 

Braat et al. 2005 [ [34]]

68/822

13/274

1,81

0,98 to 3,33

 

Brand et al. 2005 [ [22]]

29/408

15/398

1,95

1,03 to 3,70

 

Lakatos et al. 2005 [ [35]]

104/1054

48/400

0,80

0,56 to 1,15

 

Gazouli et al. 2005 [ [36]]

19/240

6/200

2,78

1,09 to 7,10

 

Oostenbrug et al. 2005 [ [37]]

53/786

27/592

1,51

0,94 to 2,43

 

Ouburg et al. 2005 [ [38]]

23/224

18/340

2,04

1,08 to 3,88

 

Fries et al. 2005 [ [39]] (1)

2/46

2/118

2,63

0,36 to 19,29

 

Fries et al. 2005 [ [39]] (2)

10/120

2/118

5,27

1,13 to 24,60

 

Zouiten-Mekki et al. 2009 [ [40]]

12/180

9/160

1,19

0,49 to 2,92

 

Hong et al. 2007 [ [41]]

26/364

32/376

0,82

0,48 to 1,42

 

Baumgart et al. 2007 [ [21]] (1)

6/288

16/404

0,51

0,20 to 1,33

 

Baumgart et al. 2007 [ [21]] (2)

28/482

49/806

0,95

0,59 to 1,54

 

Browning et al. 2007 [ [42]]

50/778

44/832

1,23

0,81 to 1,86

 

De Ridder et al. 2007 [ [23]] (1)

11/144

20/488

1,93

0,91 to 4,14

 

De Ridder et al. 2007 [ [23]] (2)

63/756

20/488

2,12

1,27 to 3,56

 

Riis et al. 2007 [ [43]]

32/422

152/1236

0,58

0,39 to 0,87

 

Hume et al. 2008 [ [44]]

87/1238

36/720

1,43

0,96 to 2,14

 

Rigoli et al. 2008 [ [45]]

10/266

8/206

0,96

0,38 to 2,49

 

Manolakis et al. 2013 [ [46]]

20/326

33/548

1,02

0,58 to 1,81

 

Current study 2014

9/166

10/224

1,22

0,49 to 3,09

 

Total (fixed effects)

825/10676

633/10152

1,26

1,13 to 1,42

0.0001

Total (random effects)

825/10676

633/10152

1,35

1,12 to 1,64

 
Table 2

Pooled analysis of studies exploring the role of TLR4 Asp299Gly in UC

Study

Cases (Events/Total)

Controls (Events/Total)

Odds ratio

95% CI

P value

Arnott et al. 2004 [ [31]]

35/492

33/378

0,801

0,49 to 1,31

 

Franchimont et al. (1) 2004 [ [32]]

32/326

14/278

2,052

1,07 to 3,93

 

Torok et al. 2004 [ [33]]

18/196

12/290

2,343

1,10 to 4,98

 

Braat et al. 2005 [ [34]]

24/452

13/274

1,126

0,56 to 2,25

 

Gazouli et al. 2005 [ [36]]

6/170

6/200

1,183

0,37 to 3,73

 

Oostenbrug et al. 2005 [ [37]]

21/358

27/592

1,304

0,72 to 2,34

 

Baumgart et al. 2007 [ [21]] (1)

8/236

16/404

0,851

0,36 to 2,02

 

Baumgart et al. 2007 [ [21]] (2)

24/290

49/806

1,394

0,84 to 2,31

 

Browning et al. 2007 [ [42]]

51/810

44/832

1,203

0,79 to 1,82

 

Riis et al. 2007 [ [43]]

53/808

152/1236

0,501

0,36 to 0,69

 

De Ridder et al. 2007 [ [23]] (1)

33/452

20/488

1,843

1,04 to 3,26

 

De Ridder et al. 2007 [ [23]] (2)

4/62

20/488

1,614

0,53 to 4,88

 

Rigoli et al. 2008 [ [45]]

3/90

8/206

0,853

0,22 to 3,29

 

Manolakis et al. 2013 [ [46]]

41/374

33/548

1,921

1,19 to 3,10

 

Current study 2014

6/68

10/224

2,071

0,72 to 5,92

 

Total (fixed effects)

359/5184

457/7244

1,092

0,94 to 1,26

0.20

Total (random effects)

359/5184

457/7244

1,268

0,95 to 1,69

 
Table 3

Pooled analysis of studies exploring the role of TLR4 Thr399Ile in CD

Study

Sample size

Cases (Events/Total)

Controls (Events/Total)

Odds ratio

95% CI

P value

Torok et al. 2004 [ [33]]

CD: 102

16/204

12/290

1.972

0.91 to 4.26

 

HC: 145

Braat et al. 2005 [ [34]]

CD: 204

30/408

19/398

1.583

0.87 to 2.86

 

HC: 199

Gazouli et al. 2005 [ [36]]

CD: 120

1/240

2/200

0.414

0.037 to 4.60

 

HC: 100

Oostenbrug et al. 2005 [ [37]]

CD: 393

69/1008

29/598

1.442

0.92 to 2.25

 

HC: 296

Zouiten-Mekki et al. 2009 [ [40]]

CD: 90

13/180

8/160

1.479

0.59 to 3.66

 

HC: 80

Hong et al. 2007 [ [41]]

CD: 182

30/364

32/376

0.966

0.57 to 1.62

 

HC: 188

Browning et al. 2007 [ [42]]

CD: 389

47/778

46/832

1.099

0.72 to 1.67

 

HC: 416

De Ridder et al. 2007 [ [23]]

CD: 450

72/900

22/488

1.842

1.12 to 3.00

 

HC: 244

Rigoli et al. 2008 [ [45]]

CD: 133

8/266

6/206

1.034

0.35 to 3.02

 

HC:103

Azzam et al. 2012 [ [47]]

CD: 46

26/92

22/100

1.397

0.72 to 2.69

 

HC: 50

Manolakis et al. 2013 [ [46]]

CD: 163

20/326

33/548

1.020

0.57 to 1.80

 

HC: 274

Our study 2014

CD: 83

7/166

3/224

3.243

0.82 to 12.73

 

HC: 112

Total (fixed effects)

 

339/4932

234/4420

1.345

1.12 to 1.60

0.002

Total (random effects)

 

339/4932

234/4420

1.336

1.11 to 1.59

Table 4

Pooled analysis of studies exploring the role of TLR4 Thr399Ile in UC

Study

Sample size

Cases (Events/Total)

Controls (Events/Total)

Odds

95% CI

P value

Torok et al. 2004 [ [33]]

UC: 98

22/196

12/290

2.929

1.41 to 6.06

 

HC: 145

Gazouli et al. 2005 [ [36]]

UC: 85

3/170

2/200

1.778

0.29 to 10.77

 

HC: 100

Oostenbrug et al. 2005 [ [37]]

UC: 179

24/434

19/598

1.784

0.96 to 3.30

 

HC: 296

Zouiten-Mekki et al. 2009 [ [40]]

UC: 30

2/60

2/160

2.724

0.37 to 19.78

 

HC: 80

Browning et al. 2007 [ [42]]

UC: 405

59/810

46/832

1.342

0.90 to 1.99

 

HC: 416

De Ridder et al. 2007 [ [23]]

UC: 257

34/514

22/488

1.500

0.86 to 2.60

 

HC: 244

Rigoli et al. 2008 [ [45]]

UC: 45

3/90

6/206

1.149

0.28 to 4.70

 

HC:103

Manolakis et al. 2013 [ [46]]

UC: 187

41/374

33/548

1.921

1.19 to 3.10

 

HC: 274

Our study 2014

UC: 34

3/68

3/224

3.400

0.67 to 17.25

 

HC: 112

Total (fixed effects)

 

191/2716

145/3546

1.695

1.35 to 2.11

0.0001

Total (random effects)

 

191/2716

145/3546

1.699

1.35 to 2.12

Table 5

Pooled analysis of studies exploring the role of TLR4 Asp299Gly in IBD

Study

Sample size

Cases (Events/Total)

Controls (Events/Total)

Odds ratio

95% CI

P value

Arnott et al. 2004 [ [31]]

IBD: 480

85/960

33/378

1,016

0,667 to 1,54

 

HC: 189

Franchimont et al. 2004 [ [32]]

IBD: 610

131/1220

14/278

2,268

1,286 to 4,00

 

HC: 139

Torok et al. 2004 [ [33]]

IBD: 200

32/400

12/290

2,014

1,019 to 3,98

 

HC: 145

Braat et al. 2005 [ [34]]

IBD: 637

92/1274

13/274

1,563

0,861 to 2,83

 

HC: 137

Gazouli et al. 2005 [ [36]]

IBD: 205

25/410

6/200

2,100

0,847 to 5,20

 

HC: 100

Oostenbrug et al. 2005 [ [37]]

IBD: 572

74/1144

27/592

1,447

0,921 to 2,27

 

HC: 296

Baumgart et al. 2007 [ [21]] (1)

IBD: 262

14/524

16/404

0,666

0,321 to 1,38

 

HC: 202

Baumgart et al. 2007 [ [21]] (2)

IBD: 386

52/772

49/806

1,116

0,745 to 1,67

 

HC: 403

Browning et al. 2007 [ [42]]

IBD: 796

101/1592

44/832

1,213

0,843 to 1,74

 

HC: 416

Riis et al. 2007 [ [43]]

IBD: 615

85/1230

152/1236

0,529

0,401 to 0,69

 

HC: 618

De Ridder et al. 2007 [ [23]]

IBD: 103

15/206

20/488

1,838

0,921 to 3,66

 

De Ridder et al. 2007 [ [23]]

IBD: 604

96/1208

20/488

2,020

1,233 to 3,31

 

HC: 244

Rigoli et al. 2008 [ [45]]

IBD: 178

13/356

8/206

0,938

0,382 to 2,30

 

HC: 103

Manolakis et al. 2013 [ [46]]

IBD: 350

61/700

33/548

1,490

0,960 to 2,31

 

HC: 274

Our study 2014

IBD:117

15/234

10/224

1,466

0,644 to 3,33

 

HC:112

Total (fixed effects)

 

891/12230

457/7244

1,154

1,021 to 1,30

0.015

Total (random effects)

 

891/12230

457/7244

1,306

1,006 to 1,69

Table 6

Pooled analysis of studies exploring the role of TLR4 Thr399Ile in IBD

Study

Sample size

Cases (Events/Total)

Controls (Events/Total)

Odds

95% CI

P value

Torok et al. 2004 [ [33]]

IBD: 200

38/400

12/290

2.432

1.24 to 4.74

 

HC: 145

Gazouli et al. 2005 [ [36]]

IBD: 205

4/410

2/200

0.975

0.17 to 5.37

 

HC: 100

Oostenbrug et al. 2005 [ [37]]

IBD: 721

93/1442

29/598

1.353

0.88 to 2.07

 

HC: 299

Zouiten-Mekki et al. 2009 [ [40]]

IBD: 120

15/240

8/160

1.267

0.52 to 3.06

 

HC: 80

Browning et al. 2007 [ [42]]

IBD: 794

106/1588

46/832

1.222

0.85 to 1.74

 

HC: 416

De Ridder et al. 2007 [ [23]]

IBD: 707

106/1414

22/488

1.717

1.07 to 2.75

 

HC: 244

Rigoli et al. 2008 [ [45]]

IBD: 178

12/356

6/206

1.163

0.43 to 3.14

 

HC: 103

Manolakis et al. 2013 [ [46]]

IBD: 350

61/700

33/548

1.490

0.96 to 2.31

 

HC: 274

Our study 2014

IBD: 117

10/234

3/224

3.289

0.89 to 12.11

 

HC: 112

Total (fixed effects)

 

445/6784

161/3546

1.479

1.22 to 1.82

0.0001

Total (random effects)

 

445/6784

161/3546

1.465

1.21 to 1.80

Results

Hundred and seventeen patients with IBD (83 CD; 34 UC) and 112 control subjects from the general population were genotyped for the presence of TLR4Asp299Gly and Thr399Ile polymorphisms.

The average age of CD, UC patients and controls was 27.6 ± 2.3, 40 ± 5.0 and 31.3 ± 2.1 years respectively. The distributions of genotype and allele frequencies of both TLR4Asp299Gly and Thr399Ile polymorphisms in CD patients (X2 = 0.03, P = 0.86; X2 = 0.02, P = 0.90) and healthy controls (X2 = 2.86, P = 0.24; X2 = 0.01, P = 0.94) were in Hardy-Weinberg equilibrium. In patients with UC, genotype and allele frequencies distributions for Asp299Gly polymorphism (X2 = 0.03, P = 0.86) were in Hardy-Weinberg equilibrium but not for Th399ILeu polymorphism (X2 = 19.05, P <0.001).

In order to study associations of TLR4 variants in IBD overall and in CD and UC in particular, the distribution of TLR4 polymorphic alleles was assessed. Genotype and allele frequencies are given in Table 7 and genotypic and allelic odds ratios and test P-values are presented in Table 8. None CD nor UC colitis patients were homozygous for G allele. Mutant allele frequency was 5.4% in CD, 8.8% in UC and 4.5% in HC. No significant difference was noticed in allele distributions of the Asp299Gly polymorphism between the control and patient groups. Likewise, no significant association of IBD with the Thr399Ile polymorphism was found in either cohort (allele frequencies: HC 1.3%, CD 4.2%, UC 4.4%). TT genotype was not observed in both CD patients and HC and only one individual carried the 399Ile variant at both alleles in UC. Co-segregation of TLR4 polymorphic alleles was observed in only 33% of controls (3 out of 9), 33% in UC (2 out of 6) and 60% in CD (6 out of 10).
Table 7

Allele and genotype frequencies of the studied polymorphisms in the group of patients with Crohn’s disease, ulcerative colitis and controls

Group

TLR4 Asp299Gly

 

TLR4 Thr399Ile

A

G

AA

AG

GG

C

T

CC

CT

TT

CD (%) N = 83

157 (94.6)

9 (5.4)

74 (89.2)

9 (10.8)

-

159 (95.8)

7 (4.2)

76 (91.6)

7 (8.4)

-

UC (%) N = 34

62 (91.2)

6 (8.8)

28 (82.4)

6 (17.6)

-

65 (95.6)

3 (4.4)

32 (94.1)

1 (2.9)

1 (2.9)

Controls (%) N = 112

214 (95.5)

10 (4.5)

103 (92.0)

8 (7.1)

1 (0.9)

221 (98.7)

3 (1.3)

109 (97.3)

3 (2.7)

-

Table 8

Odds ratios and P values for association of TLR4 variants with IBD status

SNP

Trait

Genotype/Allele

OR

CI

P Value

Asp299Gly

CD

AG

1.57

(0.58-4.25)

0.38

G

1.23

(0.49-3.09)

0.66

UC

AG

2.76

(0.88-8.61)

0.08

G

2.07

(0.72-5.92)

0.17

Thr399Ile

CD

CT

3.35

(0.84-13.35)

0.09

T

3.24

(0.83-12.74)

0.09

UC

CT

1.14

(0.11-11.29)

0.91

TT

10.11

(0.40-254.1)

0.16

T

3.4

(0.67-17.25)

0.14

Meta-analysis of our dataset with the published studies showed a significant association between TLR4 Asp299Gly variant allele and CD risk in a total of 5338 cases and 5076 controls (Pooled ORs = 1.35, 95% CI: 1.12-1.38; P = 0.0001) (Figure 1). In the other hand, no association with UC was found when evaluating disease risk in 2592 patients and 3622 controls (Table 2), OR = 1.27, 95% CI = 0.95-1.69; P = 0.20 (Figure 2). Heterogeneity in odds ratios between studies was evidenced for CD (Q = 54.5, 23 df, P = 0.0002, I2 = 57.6%) and UC (Q = 43.4%, 14 df, P = 0.0001,I2 = 67.8).
Figure 1
Figure 1

Forest plots for the association of TLR4 A299G and risk of CD.

Figure 2
Figure 2

Forest plots for the association of TLR4 A299G and risk of UC.

Combining Asp299Gly results for CD and UC (6115 cases and 3622 controls), an overall significant increased risk for IBD was observed, OR = 1.15, 95% CI = 1.03-1.30; P = 0.015 (Figure 3). However, a significant heterogeneity in allelic frequencies distribution is reported (Cohran’s Q = 52.9, I2 = 73.6%).
Figure 3
Figure 3

Forest plots for the association of TLR4 A299G and risk of IBD.

Based on the studies published so far combined to our results, we observed a significant association between the T allele of the TLR4Thr399Ile Polymorphism and both CD and UC risk (Figures 4 and 5). As well, TLR4Thr399Ile variant increased the overall IBD susceptibility when combining CD and UC results (OR = 1.46, 95%CI: 1.21-1.76; P < 0.0001) for a total of 3392 cases and 1773 controls (Figure 6).
Figure 4
Figure 4

Forest plots for the association of TLR4 T399I and risk of CD.

Figure 5
Figure 5

Forest plots for the association of TLR4 T399I and risk of UC.

Figure 6
Figure 6

Forest plots for the association of TLR4 T399I and risk of IBD.

In the present meta-analysis, we did not observed heterogeneity between studies for TLR4Thr399Ile Polymorphism distribution in CD (Q = 9.05, DF = 11, I2 = −21.54%; P = 0.62), UC (Q = 5.18, Df = 8, I2 = −54.4%; P = 0.73) and IBD (Cohran’sQ = 7.84, DF = 10, I2 = −27.5%; P = 0.64).

Genotype-phenotype correlation was investigated; demographic and clinical characteristics of CD and UC patients according to TLR4 polymorphisms are shown in (Tables 9 and 10). A significant association was found between the need for surgery and possession of one or more Asp299gly variant alleles in UC patients (P = 0.004). The presence of TLR4 variant alleles was not associated with smoking habits, age of diagnosis, disease location and behavior, family history and presence of extra-intestinal manifestations. Separate analyses in men and women did not reveal sex related associations. None of our UC patients had an appendectomy. The correlation study between Th399ILeu polymorphism and CD or UC didn’t allow to associate TLR4 genotype with a particular phenotype.
Table 9

Genotype-phenotype correlations in patients with Crohn’s disease

Parameter

N

Asp299gly (%)

P value

Chi-square test

Thr399Ile (%)

P value

Chi-square test

AA

AG

GG

CC

CT

TT

  

Age of onset

83

   

0.48

1.45

   

0.59

1.05

<17 years

10

10 (100.0)

-

   

10 (100.0)

-

   

17-40

63

55 (87.3.)

8 (12.7)

   

57 (90.5)

6 (9.5)

   

>40 years

10

9 (90.0)

1 (10.0)

   

9 (90.0)

1 (10.0)

   

Sex

83

   

0.35

0.87

   

0.17

1.92

Woman

25

24 (96.0)

1 (4.0)

   

25 (100.0)

-

   

Man

58

50 (86.2)

8 (13.8)

   

51 (87.9)

7 (12.1)

   

Type

83

   

0.54

2.14

   

0.36

3.23

Fistulizing

35

31 (88.6)

4 (11.4)

   

33 (94.3)

2 (5.7)

   

Non fistulizing non stenosing

24

20 (83.3)

4 (16.7)

   

20 (83.3)

(16.7)

   

Stenosing

17

16 (94.1)

1 (5.9)

   

16 (94.1)

1 (5.9)

   

Fistulizing stenosing

7

7 (100.0)

-

   

7 (100.0)

-

   

Localization

83

   

0.80

1.67

   

0.94

0.82

L1

30

26 (86.7)

4 (13.3)

   

27 (90.0)

3 (10.0)

   

L2

20

17 (85.0)

3 (5.0)

   

18 (90.0)

2 (10.0)

   

L3

26

24 (92.3)

2 ()

   

24 (92.3)

2 (7.7)

   

L4

3

3 (100.0)

-

   

3 (100.0)

-

   

L4 + L2

4

4 (100.0)

-

   

4 (100.0)

-

   

Smoking

83

   

0.04

4.44

   

0.17

1.92

Presence

33

26 (78.8)

7 (21.2)

   

28 (84.8)

5 (15.2)

   

Absence

50

48 (96.0)

2 (4.0)

   

48 (96)

2 (4.0)

   

SFC

83

   

0.91

0.01

   

0.76

0.09

Presence

4

4 (100.0)

-

   

4 (100.0)

-

   

Absence

79

70 (88.6)

9 (11.4)

   

72 (91.1)

7 (8.9)

   

Appendectomy

83

   

0.84

0.04

   

0.58

0.30

Presence

12

11 (91.7)

1 (8.3)

   

10 (83.3)

2 (16.7)

   

Absence

71

63 (88.7)

8 (11.3)

   

66 (93.0)

5 (7.0)

   

EIM

83

   

0.97

0.001

   

0.97

0.001

Presence

42

38 (90.5)

4 (9.5)

   

39 (92.9)

3 (7.1)

   

Absence

41

36 (87.8)

5 (12.2)

   

37 (95.1)

4 (4.9)

   

Surgery

83

   

0.50

0.45

   

0.45

0.57

Presence

41

38 (92.7)

3 (7.3)

   

39 (95.1)

2 (4.9)

   

Absence

42

36 (85.7)

6 (14.3)

   

37 (88.1)

5 (11.9)

   

SFC: similar familial cases; EIM: extra intestinal manifestations; N: total number; AA: wild type TLR4 Asp299gly, AG: TLR4 Asp299gly heterozygous variant, GG: TLR4 Asp299gly homozygous variant; CC: wild type TLR4 Thr399Ile, CT: TLR4 Thr399Ile heterozygous variant, TT: TLR4 Thr399Ile homozygous variant.

Table 10

Genotype-phenotype correlations in patients with ulcerative colitis

Parameter

N

Asp299gly (%)

P value

Chi-square test

Thr399Ile (%)

P value

Chi-square test

AA

AG

GG

CC

CT

TT

  

Age of onset

34

   

0.35

0.89

   

0.34

2.13

<17 years

-

-

-

   

-

-

   

17-40

20

18 (90.0)

2 (10.0)

-

  

19 (95.0)

1 (5.0)

-

  

>40 years

14

10 (71.4)

4 (28.6)

-

  

13 (92.9)

-

1 (7.1)

  

Sex

34

   

0.89

0.018

   

0.36

2.06

Woman

15

13 (86.7)

2 (13.3)

-

  

14 (93.3)

1 (6.7)

-

  

Man

19

15 (78.9)

4 (21.1)

-

  

18 (94.7)

-

1 (5.3)

  

Extent of the disease

34

   

0.31

3.55

   

0.16

9.34

E1

4

2 (50.0)

2 (50.0)

-

  

3 (75.0)

-

1 (25.0)

  

E2

15

13 (86.7)

2 (13.3)

-

  

15 (100.0)

-

-

  

E3

2

2 (100.0)

-

-

  

2 (100.0)

-

-

  

E4

13

11 (84.6)

2 (15.4)

-

  

12 (92.3)

1 (7.7)

-

  

SFC

34

   

0.39

0.74

   

0.97

0.06

Presence

1

1 (100.0)

-

-

  

1 (100.0)

-

   

Absence

33

27 (81.8)

6 (18.2)

-

  

31 (93.9)

1 (3.0)

1 (3.0)

  

Smoking

34

   

0.93

0.008

   

0.20

3.17

Presence

9

7 (77.8)

2 (22.2)

-

  

8 (88.9)

-

1 (11.1)

  

Absence

25

21 (84.)

4 (16.0)

-

  

24 (96.0)

1 (4.0)

-

  

EIM

34

   

0.89

0.018

   

0.35

2.05

Presence

19

15 (78.9)

4 (21.1)

-

  

18 (94.7)

-

1 (5.3)

  

Absence

15

13 (86.7)

2 (13.3)

-

  

14 (93.3)

1 (6.7)

-

  

Surgery

34

   

0.004

8.3

   

0.08

4.97

Presence

6

2 (33.3)

4 (66.7)

-

  

5 (83.3)

1 (16.7)

-

  

Absence

28

26 (92.9)

2 (7.1)

-

  

27 (96.4)

-

1 (3.6)

  

SFC: similar familial cases; EIM: extra intestinal manifestations; N: total number; AA: wild type TLR4 Asp299gly, AG: TLR4 Asp299gly heterozygous variant, GG: TLR4 Asp299gly homozygous variant; CC: wild type TLR4 Thr399Ile, CT: TLR4 Thr399Ile heterozygous variant, TT: TLR4 Thr399Ile homozygous variant.

Discussion

Given the evidence that an altered innate immune response and chronic inflammation are implicated in IBD pathogenesis, genetic influence of pattern recognition receptors was clearly suggested as a trigger of CD and UC. Several efforts were undertaken to demonstrate associations of the human TLR4 gene (Gene map locus 9q32-q33) with IBD and its clinical manifestation. Attention was focused on co-segregating SNPs located in exon 3 of TLR4 causing amino acid exchanges at positions 299 (Asp299Gly) and 399 (Thr399Ile) which are located in the extracellular domain of the receptor [17],[48]. Association of TLR4 Asp299Gly with CD was first reported by Braat et al. [30] subsequent studies have had divergent results and showed strong evidence of ethnic differences. In view of the discrepant data regarding the association of the TLR4 gene with IBD and its clinical complications, we investigated for the first time the potential influence of TLR4 SNPs in the susceptibility to IBD in a cohort of Moroccan patients. However, the statistical power was very low and could be considered a limitation in this study. Our study showed that the GG genotype was not found in both CD and UC patients. No significant differences were observed in allele frequencies of the TLR4Asp299Gly among patients and controls. In addition, although slightly increased frequencies of the mutant alleles were encountered, we were not able to identify a significant difference in allele distributions of the TLR4Thr399Ile in our case–control study. In line with our results, a Tunisian study that genotyped 90 patients with CD and 80 healthy individuals for the Asp299Gly and Thr399Ile polymorphisms, reported the absence of association between CD and TLR4 gene in a north African population [40]. Although the Tunisian CD population showed a similar overall pattern of allelic frequencies, it is of some note that the genotype-phenotype correlation revealed divergent results. While the Thr399Ile variant allele was associated with early disease onset in Tunisian patients, no correlation with a particular phenotype was observed for this polymorphism in the Moroccan patients. Our study showed that the presence of Asp299Gly variant allele was associated with the need for surgery in UC patients (P = 0.004). Furthermore, the occurrence of one Asp299gly risk allele in CD patients was suggestive of a trend of association with smoking habits (P = 0.04) that was no more observed after correction for multiple testing.

Being in linkage disequilibrium, TLR4 mutant alleles are known to be inherited in the form of Asp299Gly/Thr399Ile haplotype [48]. In a German cohort, the co-segregation between mutant alleles represented 100% in controls, whereas it was not complete in CD and UC patients: 94% and 86% respectively [33]. These observations contrast our findings where simultaneous presence of the mutated alleles was only observed in 33% of controls, 33% of UC and 60% of CD patients.

Results on the relationship of Asp299Gly SNP alone or in combination with Thr399Ile with IBD are inconsistent between studies. No difference in TLR4 allele frequency between IBD patients and controls was observed in Hungarian [35], Saudi Arabian [47], Southern Italian [45], New Zealandian [41] and EC-IBD [43] study groups populations. Genetic heterogeneity within Europe was evidenced by Arnott et al. when reporting lack of association of TLR4 and CD14 variants in Scottish and Irish CD patients [31]. Moreover, Baumgart DC et al. reported an association between IBD and the CD14 c.1-260C T promoter but not with the TLR4 (p.D299G) variant in Germans and Hungarians [21]. Interestingly, the heterozygous and homozygous pattern for the mutated allele was not detected in any of the individuals from the Japanese [49], Korean [24], Chinese Han population [25] and Zhuang population from the Guangxi Zhuang Autonomous Region of China [26]. TLR4 was linked to an increased IBD (CD or UC) risk in many other diverse investigations. Significant associations were found in patients drawn from Belgian [32], German [22],[33], Greek [36],[46] and Dutch [34] populations. In addition, several meta-analyses provided evidence that the Asp299Gly SNP is associated with CD and IBD in Caucasians [27],[42],[50]. The results of our meta-analysis indicate a significant association between TLR4 Thr399Ile and CD and UC risk in different populations. Noteworthy, a lack of heterogeneity between studies was observed regarding distribution of this polymorphism. The results support that this variant is a potential risk factor for IBD.

A correlation between Asp299Gly variant and an increased disease risk is also reported for CD and IBD but not regarding UC, which is in line with de Jager et al. findings [28]. This indicates that the risk allele is either not associated with disease susceptibility or that the small number of UC cohorts did not provide sufficient power to detect an association. A correlation between TLR4 polymorphisms and UC has been rarely discussed, more association studies are needed to validate the conclusion.

To date it is well known that the frequency of the investigated TLR4 gene SNPs varies between populations [27],[31]. Overall, there was inescapable evidence for considerable genetic heterogeneity. This observation has been explained by geographic and ethnicity-related gene effect on disease susceptibility [51]. Our results showed that the distribution of the risk alleles varies between both TLR4 polymorphisms. Therefore, we offer additional evidence for differences in the contribution of individual genetic determinants between populations. Browning et al., argued that negative studies with results that do not achieve statistical significance can still contribute evidence for association, having important implications for the first generation of whole genome association studies [42].

In view of the role of potential confounders related to the present study and to discrepant results between populations, it is likely that the contribution of different sample size, selection bias, phenotypic heterogeneity and population stratification in case control studies can’t be ruled out. These data demonstrate further the real difficulties in candidate gene analysis in complex diseases. Moreover, given that IBD is a polygenic disease it is provided that association studies will reveal various sets of susceptible genes. Therefore, further large-scale studies are required to obtain a clear insight into the impact of the pattern recognition receptors in the pathophysiological and immunogenetic aspects of IBD and to explore the contribution of other genes involved in various processes.

Conclusions

In the present study, we have demonstrated that the common mutations in the TLR4 gene are not associated with IBD in a sample of Moroccan patients. However, our dataset contributed to the significant association observed in TLR4 meta-analysis.

It is likely that the distribution of TLR4 gene polymorphisms have ethnic differences. Our data suggests that other genetic and environmental factors may play a role in IBD susceptibility and behavior in this population. However, because of the relatively small sample size, additional well-powered studies are needed to confirm our findings.

Abbreviations

IBD: 

Inflammatory bowel disease

CD: 

Crohn’s disease

UC: 

Ulcerative colitis

PRR: 

Pattern recognition receptors

Declarations

Acknowledgements

We gratefully acknowledge the members of LGMP and the Gastroenterology department. We are also indebted to Mr. Wifaq Said, Center of Doctoral studies, for his help and for the secretarial assistance.

Authors’ Affiliations

(1)
Laboratory of Genetic and Molecular Pathology (LGPM), Medical School, Hassan II University, Casablanca, Morocco
(2)
Gastroenterology Department, CHU Ibn Rochd, Casablanca, Morocco
(3)
Laboratory of Thrombosis and Haemostasis Research Centre, Montreal Heart Institute, 5000 Belanger Street, Montreal, H1T 1C8, QC, Canada

References

  1. Shanahan F, Bernstein CN: The evolving epidemiology of inflammatory bowel disease. Curr Opin Gastroenterol. 2009, 25 (4): 301-305. 10.1097/MOG.0b013e32832b12ef.View ArticlePubMedGoogle Scholar
  2. Sobczak M, Fabisiak A, Murawska N, Wesołowska E, Wierzbicka P, Wlazłowski M, Wójcikowska M, Zatorski H, Zwolińska M, Fichna J: Current overview of extrinsic and intrinsic factors in etiology and progression of inflammatory bowel diseases. Pharmacol Rep. 2014, 66 (5): 766-775. 10.1016/j.pharep.2014.04.005.View ArticlePubMedGoogle Scholar
  3. Rock FL, Hardiman G, Timans JC, Kastelein RA, Bazan JF: A family of human receptors structurally related to Drosophila Toll. Proc Natl Acad Sci U S A. 1998, 95: 588-593. 10.1073/pnas.95.2.588.View ArticlePubMedPubMed CentralGoogle Scholar
  4. Takeda K, Kaisho T, Akira S: Toll-like receptors. Annu Rev Immunol. 2003, 21: 335-376. 10.1146/annurev.immunol.21.120601.141126.View ArticlePubMedGoogle Scholar
  5. Kumar H, Kawai T, Akira S: Toll-like receptors and innate immunity. Biochem Biophys Res Commun. 2009, 388: 621-625. 10.1016/j.bbrc.2009.08.062.View ArticlePubMedGoogle Scholar
  6. Medzhitov R, Preston-Hurlburt P, Janeway CA: A human homologue of the Drosophila Toll protein signals activation of adaptive immunity. Nature. 1997, 388 (6640): 394-397. 10.1038/41131.View ArticlePubMedGoogle Scholar
  7. Akira S, Takeda K, Kaisho T: Toll-like receptors: critical proteins linking innate and acquired immunity. Nat Immunol. 2001, 2: 675-680. 10.1038/90609.View ArticlePubMedGoogle Scholar
  8. Chow JC, Young DW, Golenbock DT, Christ WJ, Gusovsky F: Toll-like receptor-4 mediates lipopolysaccharide-induced signal transduction. J Biol Chem. 1999, 274: 10689-10692. 10.1074/jbc.274.16.10689.View ArticlePubMedGoogle Scholar
  9. Fitzgerald KA, Palsson McDermott EM, Bowie AG, Jefferies CA, Mansell AS, Brady G, Brint E, Dunne A, Gray P, Harte MT, McMurray D, Smith DE, Sims JE, Bird TA, O'Neill LA: Mal (MyD88-adapter-like) is required for Tolllike receptor-4 signal transduction. Nature. 2001, 413 (6851): 78-83. 10.1038/35092578.View ArticlePubMedGoogle Scholar
  10. Otte JM, Cario E, Podolsky DK: Mechanisms of cross hyporesponsiveness to Toll-like receptor bacterial ligands in intestinal epithelial cells. Gastroenterology. 2004, 126: 1054-1070. 10.1053/j.gastro.2004.01.007.View ArticlePubMedGoogle Scholar
  11. Abreu MT, Vora P, Faure E, Thomas LS, Arnold ET, Arditi M: Decreased expression of Toll-like receptor- 4 and MD-2 correlates with intestinal epithelial cell protection against dysregulated proinflammatory gene expression in response to bacterial lipopolysaccharide. J Immunol. 2001, 167: 1609-1616. 10.4049/jimmunol.167.3.1609.View ArticlePubMedGoogle Scholar
  12. Cario E, Podosky DK: Differential alteration in intestinal epithelial cell expression of Toll-like receptor 3 (TLR3) and TLR4 in inflammatory bowel disease. Infect Immun. 2000, 68: 7010-7017. 10.1128/IAI.68.12.7010-7017.2000.View ArticlePubMedPubMed CentralGoogle Scholar
  13. Hausmann M, Kiessling S, Mestermann S, Webb G, Spöttl T, Andus T, Schölmerich J, Herfarth H, Ray K, Falk W, Rogler G: Toll-like receptors 2 and 4 are up-regulated during intestinal inflammation. Gastroenterology. 2002, 122: 1987-2000. 10.1053/gast.2002.33662.View ArticlePubMedGoogle Scholar
  14. Singh JC, Cruickshank SM, Newton DJ, Wakenshaw L, Graham A, Lan J, Lodge JP, Felsburg PJ, Carding SR: Toll-like receptor mediated responses of primary intestinal epithelial cells during the development of colitis. Am J PhysiolGastrointest Liver Physiol. 2005, 288: G514-G524. 10.1152/ajpgi.00377.2004.View ArticleGoogle Scholar
  15. Frolova L, Drastich P, Rossmann P, Klimesova K, Tlaskalova-Hogenova H: Expression of Toll-like receptor 2 (TLR2), TLR4, and CD14 in biopsy samples of patients with inflammatory bowel diseases: upregulated expression of TLR2 in terminal ileum of patients with ulcerative colitis. J Histochem Cytochem. 2008, 56: 267-274. 10.1369/jhc.7A7303.2007.View ArticlePubMedPubMed CentralGoogle Scholar
  16. Szebeni B, Veres G, Dezsõfi A, Rusai K, Vannay A, Mraz M, Majorova E, Arató A: Increased expression of Toll-like receptor (TLR) 2 and TLR4 in the colonic mucosa of children with inflammatory bowel disease. Clin Exp Immunol. 2008, 151: 34-41. 10.1111/j.1365-2249.2007.03531.x.View ArticlePubMedPubMed CentralGoogle Scholar
  17. Arbour NC, Lorenz E, Schutte BC: TLR4 mutations are associated with endotoxin hyporesponsiveness in humans. Nat Genet. 2000, 25: 187-191. 10.1038/76048.View ArticlePubMedGoogle Scholar
  18. Senhaji N, Serbati N, Diakité B, Arazzakou S, Hamzi K, Badre W, Nadifi S: Methylenetetrahydrofolatereductase C677T variant in Moroccan patients with inflammatory bowel disease. Gene. 2013, 521 (1): 45-49. 10.1016/j.gene.2013.02.046.View ArticlePubMedGoogle Scholar
  19. Serbati N, Senhaji N, Diakite B, Badre W, Nadifi S: IL23R and ATG16L1 variants in Moroccan patients with inflammatory bowel disease. BMC Res Notes. 2014, 7 (1): 570-10.1186/1756-0500-7-570.View ArticlePubMedPubMed CentralGoogle Scholar
  20. Silverberg MS, Satsangi J, Ahmad T, Arnott ID, Bernstein CN, Brant SR, Caprilli R, Colombel JF, Gasche C, Geboes K, Jewell DP, Karban A, Loftus EV, Peña AS, Riddell RH, Sachar DB, Schreiber S, Steinhart AH, Targan SR, Vermeire S, Warren BF: Toward an integrated clinical, molecular and serological classification of inflammatory bowel disease: report of a working party of the 2005 Montreal World Congress of Gastroenterology. Can J Gastroenterol. 2005, 19: 5-36.View ArticleGoogle Scholar
  21. Baumgart DC, Buning C, Geerdts L, Schmidt HH, Genschel J, Fiedler T, Gentz E, Molnar T, Nagy F, Lonovics J, Lochs H, Wiedenmann B, Nickel R, Witt H, Dignass A: The c.1–260C > T promoter variant of CD14 but not the c.896A > G (p.D299G) variant of toll-like receptor 4 (TLR4) genes is associated with inflammatory bowel disease. Digestion. 2007, 76: 196-202. 10.1159/000112646.View ArticlePubMedGoogle Scholar
  22. Brand S, Staudinger T, Schnitzler F, Pfennig S, Hofbauer K, Dambacher J, Seiderer J, Tillack C, Konrad A, Crispin A, Goke B, Lohse P, Ochsenkuhn T: The role of Toll-like receptor 4 Asp299Gly and Thr399Ile polymorphisms and CARD15/NOD2 mutations in the susceptibility and phenotype of Crohn’s disease. Inflamm Bowel Dis. 2005, 11: 645-652. 10.1097/01.MIB.0000168372.94907.d2.View ArticlePubMedGoogle Scholar
  23. de Ridder L, Weersma RK, Dijkstra G, van der Steege G, Benninga MA, Nolte IM, Taminiau JA, Hommes DW, Stokkers PC: Genetic susceptibility has a more important role in pediatric-onset Crohn’s disease than in adult-onset Crohn’s disease. Inflamm Bowel Dis. 2007, 13: 1083-1092. 10.1002/ibd.20171.View ArticlePubMedGoogle Scholar
  24. Kim EJ, Chung WC, Lee KM, Paik CN, Jung SH, Lee BI, Chae HS, Choi KY: Association between toll-like receptors/CD14 gene polymorphisms and inflammatory bowel disease in Korean population. J Korean Med Sci. 2012, 27: 72-77. 10.3346/jkms.2012.27.1.72.View ArticlePubMedGoogle Scholar
  25. Guo QS, Xia B, Jiang Y, Morre SA, Cheng L, Li J, Crusius JB, Pena AS: Polymorphisms of CD14 gene and TLR4 gene are not associated with ulcerative colitis in Chinese patients. Postgrad Med J. 2005, 81: 526-529. 10.1136/pgmj.2004.030825.View ArticlePubMedPubMed CentralGoogle Scholar
  26. Chen L, Lin MJ, Zhan LL, Lv XP: Analysis of TLR4 and TLR2 polymorphisms in inflammatory bowel disease in a Guangxi Zhuang population. World J Gastroenterol. 2012, 18: 6856-6860. 10.3748/wjg.v18.i46.6856.View ArticlePubMedPubMed CentralGoogle Scholar
  27. Shen X, Shi R, Zhang H, Li K, Zhao Y, Zhang R: The Toll-like receptor 4 D299G and T399I polymorphisms are associated with Crohn’s disease and ulcerative colitis: a meta-analysis. Digestion. 2010, 81 (2): 69-77. 10.1159/000260417.View ArticlePubMedGoogle Scholar
  28. De Jager PL, Franchimont D, Waliszewska A, Bitton A, Cohen A, Langelier D, Belaiche J, Vermeire S, Farwell L, Goris A, Libioulle C, Jani N, Dassopoulos T, Bromfield GP, Dubois B, Cho JH, Brant SR, Duerr RH, Yang H, Rotter JI, Silverberg MS, Steinhart AH, Daly MJ, Podolsky DK, Louis E, Hafler DA, Rioux JD: The role of the Toll receptor pathway in susceptibility to inflammatory bowel diseases. Genes Immun. 2007, 8: 387-397. 10.1038/sj.gene.6364398.View ArticlePubMedGoogle Scholar
  29. Figueroa C, Peralta A, Herrera L, Castro P, Gutierrez A, Valenzuela J, Aguillon JC, Quera A, Hermoso MA: NOD2/CARD15 and Toll-like 4 receptor gene polymorphism in Chilean patientes with inflammatory bowel disease. Eur Cytokine Netw. 2006, 17: 125.1340-Google Scholar
  30. Braat H, Dijgraaf M, Curvers W, Vogels E, Bodegraven AV, Stokkers P, Hommes D, Deventer SV: A functional single polymorphism of the TLR4 gene is correlated Crohn’s disease but not with ulcerative colitis. Gastroenterology. 2003, 124: A367-10.1016/S0016-5085(03)81859-0.View ArticleGoogle Scholar
  31. Arnott IDR, Nimmo ER, Drummond HE, Fennell J, Smith BR, MacKinlay E, Morecroft J, Anderson N, Kelleher D, O'Sullivan M, McManus R, Satsangi J: NOD2/CARD15, TLR4 and CD14 mutations in Scottish and Irish Crohn’s disease patients: evidence for genetic heterogeneity within Europe?. Genes Immun. 2004, 5: 417-425. 10.1038/sj.gene.6364111.View ArticlePubMedGoogle Scholar
  32. Franchimont D, Vermeir S, El Housni H, Pierik M, Van Steen K, Gustot T, Quertinmont E, Abramowicz M, Van Gossum A, Devière J, Rutgeerts P: Deficient host-bacteria interactions in inflammatory bowel disease? The toll-like receptor (TLR)-4 Asp 299Gly polymorphism is associated with Crohn’s disease and ulcerative colitis. Gut. 2004, 53: 987-992. 10.1136/gut.2003.030205.View ArticlePubMedPubMed CentralGoogle Scholar
  33. Torok HP, Glas J, Tonenchi L, Mussack T, Folwaczny C: Polymorphisms of the lipopolysaccharide-signaling complex in inflammatory bowel disease: association of a mutation in the toll-like receptor 4 gene with ulcerative colitis. Clin Immunol. 2004, 112: 85-91. 10.1016/j.clim.2004.03.002.View ArticlePubMedGoogle Scholar
  34. Braat H, Stokkers P, Hommes T, Cohn D, Vogels E, Pronk I, Spek A, van Kampen A, van Deventer S, Peppelenbosch M, Hommes D: Consequence of functional Nod2 and Tlr4 mutations on gene transcription in Crohn’s disease patients. J Mol Med (Berl). 2005, 83 (8): 601-609. 10.1007/s00109-005-0685-x.View ArticleGoogle Scholar
  35. Lakatos PL, Lakatos L, Szalay F, Willheim-Polli C, Österreicher C, Tulassay Z, Molnar T, Reinisch W, Papp J, Mozsik G, Ferenci P, Hungarian IBD Study Group: Toll-like receptor 4 and NOD2/CARD15 mutations in Hungarian patients with Crohn’s disease: Phenotype-genotype correlations. World J Gastroenterol. 2005, 11: 1489-1495. 10.3748/wjg.v11.i10.1489.View ArticlePubMedPubMed CentralGoogle Scholar
  36. Gazouli M, Mantzaris G, Kotsinas A, Zacharatos P, Papalambros E, Archimandritis E, Ikonomopoulos J, Gorgoulis VG: Association between polymorphisms in the Toll-like receptor 4, CD14 and CARD15/NOD2 and inflammatory bowel disease in the Greek population. W J Gastroenterol. 2005, 11: 681-685. 10.3748/wjg.v11.i5.681.View ArticleGoogle Scholar
  37. Oostenbrug LE, Drenth JP, de Jong DJ, Nolte IM, Oosterom E, van Dullemen HM, van der Linde K, te Meerman GJ, van der Steege G, Kleibeuker JH, Jansen PL: Association between Toll‐like receptor 4 and inflammatory bowel disease. Inflamm Bowel Dis. 2005, 11 (6): 567-575. 10.1097/01.MIB.0000161305.81198.0f.View ArticlePubMedGoogle Scholar
  38. Ouburg S, Mallant-Hent R, Crusius JBA, van Bodegraven AA, Mulder CJJ, Linskens R, Pena AS, Morre SA: The toll-like receptor 4 (TLR4) Asp299Gly polymorphism is associated with colonic localisation of Crohn’s disease without a major role for the Saccharomyces cerevisiae mannan-LBP-CD14-TLR4 pathway. Gut. 2005, 54 (3): 439-440.PubMedPubMed CentralGoogle Scholar
  39. Fries W, Renda MC, Lo Presti MA, Raso A, Orlando A, Oliva L, Giofré MR, Maggio A, Mattaliano A, Macaluso A, Cottone M: Intestinal permeability and genetic determinants in patients, first-degree relatives, and controls in a high-incidence area of Crohn’s disease in southern Italy. Am J Gastroenterol. 2005, 100: 2730-2736. 10.1111/j.1572-0241.2005.00325.x.View ArticlePubMedGoogle Scholar
  40. Zouiten-Mekki L, Kharrat M, Karoui S, Serghimi M, Fekih M, Matri S, Kallel L, Boubaker J, Filali A, Chaabouni H: Tolllike receptor 4 (TLR4) polymorphisms in Tunisian patients with Crohn’s disease: genotype-phenotype correlation. BMC Gastroenterol. 2009, 9: 62-10.1186/1471-230X-9-62.View ArticlePubMedPubMed CentralGoogle Scholar
  41. Hong J, Leung E, Fraser AG, Merriman TR, Vishnu P, Krissansen GW: TLR2, TLR4 and TLR9 polymorphisms and Crohn’s disease in a New Zealand Caucasian cohort. J Gastroenterol Hepatol. 2007, 22 (11): 1760-1766. 10.1111/j.1440-1746.2006.04727.x.View ArticlePubMedGoogle Scholar
  42. Browning BL, Huebner C, Petermann I, Gearry RB, Barclay ML, Shelling AN, Ferguson LR: Has toll-like receptor 4 been prematurely dismissed as an inflammatory bowel disease gene? Association study combined with meta-analysis shows strong evidence for association. Am J Gastroenterol. 2007, 102: 2504-2512. 10.1111/j.1572-0241.2007.01463.x.View ArticlePubMedGoogle Scholar
  43. Riis L, Ida V, Severine V, Frank W, Kostas K, Patrizia P, João F, Mouzas IA, O'Morain C, Ruiz-Ochoa V, Odes S, Binder V, Munkholm P, Moum B, Stockbrügger R, Langholz E, European Collaborative Study Group on Inflammatory Bowel Disease: The prevalence of genetic and serologic markers in an unselected European population‐based cohort of IBD patients. Inflamm Bowel Dis. 2007, 13 (1): 24-32. 10.1002/ibd.20047.View ArticlePubMedGoogle Scholar
  44. Hume GE, Fowler EV, Doecke J, Simms LA, Huang N, Palmieri O, Griffiths LR, Florin TH, Annese V, Radford-Smith GL: Novel NOD2 haplotype strengthens the association between TLR4 Asp299Gly and Crohn’s disease in an Australian population. Inflamm Bowel Dis. 2008, 14: 585-590. 10.1002/ibd.20362.View ArticlePubMedGoogle Scholar
  45. Rigoli L, Romano C, Caruso RA, Lo Presti MA, Di Bella C, Procopio V, Lo Giudice G, Amorini M, Costantino G, Sergi MD, Cuppari C, Calabro GE, Gallizzi R, Salpietro CD, Fries W: Clinical significance of NOD2/CARD15 and Toll-like receptor 4 gene single nucleotide polymorphisms in inflammatory bowel disease. World J Gastroenterol. 2008, 14: 4454-4461. 10.3748/wjg.14.4454.View ArticlePubMedPubMed CentralGoogle Scholar
  46. Manolakis CA, Kapsoritakis NA, Kapsoritaki A, Tiaka EK, Oikonomou AK, Lotis V, Vamvakopoulou D, Davidi I, Vamvakopoulos N, Potamianos SP: Readressing the role of toll-like receptor-4 alleles in inflammatory bowel disease: colitis, smoking, and seroreactivity. Dig Dis Sci. 2013, 58 (2): 371-380.PubMedGoogle Scholar
  47. Azzam N, Nounou H, Alharbi O, Aljebreen A, Shalaby M: CARD15/NOD2, CD14 and toll-like 4 receptor gene polymorphisms in Saudi patients with Crohn’s disease. Int J Mol Sci. 2012, 13: 4268-4280. 10.3390/ijms13044268.View ArticlePubMedPubMed CentralGoogle Scholar
  48. Ferwerda B, McCall MB, Verheijen K, Kullberg BJ, van der Ven AJ, Van der Meer JW, Netea MG: Functional consequences of toll-like receptor 4 polymorphisms. Mol Med. 2008, 14: 346-352. 10.2119/2007-00135.Ferwerda.View ArticlePubMedPubMed CentralGoogle Scholar
  49. Okayama N, Fujimura K, Suehiro Y, Hamanaka Y, Fujiwara M, Matsubara T, Maekawa T, Hazama S, Oka M, Nohara H, Kayano K, Okita K, Hinoda Y: Simple genotype analysis of the Asp299Gly polymorphism of the toll-like receptor- 4 gene that is associated with lipopolysaccharide hyporesponsiveness. J Clin Lab Anal. 2002, 16: 56-58. 10.1002/jcla.2075.View ArticlePubMedGoogle Scholar
  50. Shen XY, Shi RH, Wang Y, Zhang HJ, Zhou XQ, Shen FC, Li KB: Toll-like receptor gene polymorphisms and susceptibility to inflammatory bowel disease in Chinese Han and Caucasian populations. Zhonghua Yi XueZaZhi. 2010, 90 (20): 1416-1420.Google Scholar
  51. Ali S, Tamboli CP: Advances in epidemiology and diagnosis of inflammatory bowel diseases. Cur Gastroenterol Rep. 2008, 10: 576-584. 10.1007/s11894-008-0105-9.View ArticleGoogle Scholar

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