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• Reply to Franchimont et al
  Servaas A Morré
  Published on: 18 April 2016
• Reply:
  Denis Franchimont
  Published on: 18 April 2016
• The toll like receptor-4 gene in IBD: further evidence for genetic heterogeneity in Europe
  Ian DR Arnott
  Published on: 18 April 2016

• Published on: 18 April 2016

  Reply to Franchimont et al

  • Servaas A Morré, Laboratory of Immunogenetics
  • Other Contributors:
    • Sander Ouburg, Rosalie Mallant-Hent, J. Bart A. Crusius, Ad A van Bodegraven, Chris J.J. Mulder, Ronald Linskens and A. Salvador Peña.

  Dear Editor

  It is with great interest that we read the paper by Franchimont et al[1] in which they describe a novel association of the Toll-like receptor 4 (TLR4) +896 A>G polymorphism with both Crohn’s disease (CD) and ulcerative colitis (UC), supporting the genetic influence of pattern recognition receptors (PRRs) in triggering inflammatory bowel disease (IBD). PRRs are sensors of pattern associated molecu...

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  Dear Editor

  It is with great interest that we read the paper by Franchimont et al[1] in which they describe a novel association of the Toll-like receptor 4 (TLR4) +896 A>G polymorphism with both Crohn’s disease (CD) and ulcerative colitis (UC), supporting the genetic influence of pattern recognition receptors (PRRs) in triggering inflammatory bowel disease (IBD). PRRs are sensors of pattern associated molecular patterns (PAMPs) of microorganisms in the intestinal flora. Independently, we performed a similar study. However, special attention to the presence of anti- Saccharomyces cerevisiae antibody (ASCA) was taken since Tada et al[2] have recently reported that the S. cerevisiae mannan-LBP complex is recognized by CD14 on monocytes and signalling through TLR4 leads to the production of proinflammatory cytokines in a manner similar to that induced by LPS.

  Patients and controls were recruited from the Outpatient Department of Gastroenterology of the VU University Medical Centre, Amsterdam, The Netherlands. The group consisted of 112 CD patients and 170 unrelated Dutch Caucasian controls. Diagnosis of disease was based on clinical, histopathologic and endoscopic findings. CD patients were categorized using the Vienna classification (general patient characteristics are described elsewhere.[3] ASCA IgA and IgG ELISAs were performed as described previously.[4] Genotyping for the CD14 -260 C>T and TLR4 +896 A>G single nucleotide polymorphisms (SNPs) was performed as we described previously.[5] The CD14 -260 and TLR4 +896 genotypes, allele, and carrier frequencies were compared between the different clinical patient groups and controls. In addition, synergism between CD14 and TLR4 genotypes and alleles (carrier trait analyses) were studied. Vienna classification and ASCA status were included in the statistical modelling.

  The results are shown in Table 1.

  			CD14 –260 C>T			TLR4 +896 A>G
  			1.1 (CC)	1.2 (CT)	2.2 (TT)	1.1 (AA)	1.2 (AG)	2.2 (GG)
  Group	Vienna Classification	ntotal	n (%)	n (%)	n (%)	n (%)	n (%)	n (%)
  HC		170	48 (28)	82 (48)	40 (24)	153 (90)	16 (9)	1 (1)
  CD		112	35 (31)	54 (48)	23 (21)	91 (81)	19 (17)	2 (2)a
  CD	A1	97	29 (30)	49 (51)	19 (20)	77 (79)	19 (20)	1 (0)
  	A2	15	6 (40)	5 (33)	4 (27)	14 (93)	0 (0)	1 (6)
  	B1	43	14 (33)	20 (47)	9 (21)	36 (84)	6 (14)	1 (2)
  	B2	45	15 (33)	21 (47)	9 (20)	37 (82)	7 (16)	1 (2)
  	B3	24	6 (25)	13 (54)	5 (21)	18 (75)	6 (25)	0 (0)
  	L1	41	14 (34)	17 (41)	10 (24)	36 (88)	4 (10)	1 (2)
  	L2	23	6 (26)	13 (57)	4 (17)	13 (57)	9 (39)	1 (4)b
  	L3	47	15 (32)	23 (49)	9 (19)	41 (87)	6 (13)	0 (0)
  	L4	1	0 (0)	1 (100)	0 (0)	1 (100)	0 (0)	0 (0)

  Table 1:CD14-260 and TLR4+896 genotype distribution in CD patients and controls
  ^aTLR4*G more frequent in CD patients compared to HC (19% vs. 10%, p: 0.0489, OR: 2.076, 95% CI: 1.041 – 4.142)
  ^bTLR4*G significantly associated with colonic localization compared to non-colonic localization (43% vs. 12%; p: 0.0017, OR: 5.455, 95% CI: 1.931 – 15.410)
  Vienna Classification: [Age] A1: Age > 40y; A2: Age > 40y; [Behaviour] B1: non-stricturing, non-penetrating; B2: Stricturing; B3: Penetrating; [Localization] L1: Ileal; L2: Colonic; L3: Ileocolonic; L4: Upper GI tract;

  The frequency of the G allele of TLR4+896 was significantly increased in CD patients compared to controls (19% vs. 10%; p: 0.049; OR 2.1 [95% CI: 1.0-4.1]). Disease phenotype was assessed in the patients using the Vienna Classification. Carriage of TLR4 +896*G significantly increases the risk for colonic localization of CD compared to non-colonic localization (43% vs 12%; p: 0.0017; OR: 5.5 [95% CI: 1.9 – 15.4]). There was a clear trend (Test for trend: Chi-square: 16, p<_0.0001 when="when" we="we" compared="compared" the="the" increasing="increasing" frequency="frequency" of="of" g="g" allele="allele" tlr4="tlr4" _896="_896" in="in" controls="controls" _10="_10" to="to" cd="cd" patients="patients" _19="_19" with="with" a="a" colonic="colonic" localization="localization" _43.="_43." p="p"/> We also assessed if ASCA status was correlated with carriage of the TLR4 G allele. However, there was no difference between TLR4 G allele carriage in the ASCA positive and ASCA negative patients (23% vs 14%; p: 0.33) and there was no difference between TLR4 G allele carriage in the ASCA positive and negative CD patients with colonic localization (40% vs 46%; p: 1.00), while the frequency of G allele carriage was identical to that of CD patients with colonic localization (43%) without correcting for the ASCA status.

  Several studies have described both TLR4+896 A>G and CD14–260 C>T in CD. Klein and colleagues have described a German population and found an increased incidence of CD14 –260 heterozygous and homozygous mutants in CD patients compared to healthy controls.[6] This association could not be confirmed in our population. Preliminary data by Braat et al demonstrated an increased risk of suffering from CD in a Dutch population carrying the TLR4 +896 SNP[7] confirming our results. Franchimont et al corroborated the results from Braat et al.[1] In contrast to Franchimont et al we found a clear association between the G allele of TLR4+896 and disease phenotype (colonic localization). In contrast to the aforementioned studies and results, Arnott et al were unable to demonstrate an association between CD and the TLR4 and CD14 SNPs in a Scottish and an Irish population.[8]

  The association between TLR4 and CD underscores the role of impaired innate immunity in CD. TLR4 signalling is based on both exogenous (eg LPS) and endogenous (eg human HSPs) agonists, and since heterozygous carriership of the TLR4 +896 A>G does not seem to impair LPS signalling[9, 10] further agonist identification to elucidate the microorganisms involved in CD and especially in the colonic localization is essential to get insight in both pathophysiological and immunogenetic aspects of CD. This insight is potentially helpful to develop strategies for prevention and treatment of Crohn’s Disease.

  The association we demonstrate between TLR4 and CD is most likely not strongly based on the S. cerevisiae mannan-LBP-CD14-TLR4 pathway, as we have shown based on the ASCA data in our group. It would be interesting to know whether or not Franchimont et al tested for ASCA in their CD patients and whether or not an association between ASCA and TLR4 was observed.

  References

  (1) Franchimont D, Vermeire S, El Housni H, Pierik M, Van Steen K, Gustot T et al. Deficient host-bacteria interactions in inflammatory bowel disease? The toll-like receptor (TLR)-4 Asp299gly polymorphism is associated with Crohn's disease and ulcerative colitis. Gut 2004; 53[7]:987-992.

  (2) Tada H, Nemoto E, Shimauchi H, Watanabe T, Mikami T, Matsumoto T et al. Saccharomyces cerevisiae- and Candida albicans-derived mannan induced production of tumor necrosis factor alpha by human monocytes in a CD14- and Toll-like receptor 4-dependent manner. Microbiology and Immunology 2002;46[7]: 503-512.

  (3) Linskens RK, Mallant-Hent RC, Murillo LS, von Blomberg BM, Alizadeh BZ, Peña AS. Genetic and serological markers to identify phenotypic subgroups in a Dutch Crohn' s disease population. Dig Liver Dis 2004;36[1]: 29-34.

  (4) Linskens RK, Mallant-Hent RC, Groothuismink ZMA, Bakker-Jonges LE, van de Merwe JP, Hooijkaas H et al. Evaluation of serological markers to differentiate between ulcerative colitis and Crohn's disease: pANCA, ASCA and agglutinating antibodies to anaerobic coccoid rods. European Journal of Gastroenterology and Hepatology 2002;14[9]:1013-1018.

  (5) van der Paardt M, Crusius JBA, De Koning MHMT, Morré SA, van de Stadt RJ, Dijkmans BAC et al. No evidence for involvement of the toll-like receptor 4 (TLR4) A896G and CD14 C-260T polymorphisms in the susceptibility to ankylosing spondylitis. Ann Rheum Dis. In press.

  (6) Klein W, Tromm A, Griga T, Fricke H, Folwaczny C, Hocke M et al. A polymorphism in the CD14 gene is associated with Crohn disease. Scandinavian Journal of Gastroenterology 2002;37[2]:189-191.

  (7) Braat H, Dijkgraaf M, Curvers W, Vogels E, van Bodegraven AA, Stokkers P et al. A functional single polymorphism of the TLR4 gene is correlated with Crohn's Disease but not with Ulceratice Colitis. Gastroenterology 2004;124: A367.

  (8) Arnott IDR, Nimmo ER, Drummond HE, Fennell J, Smith BRK, MacKinlay E et al. NOD2/CARD15, TLR4 and CD14 mutations in Scottish and Irish Crohn's disease patients: evidence for genetic heterogeneity within Europe? Genes & Immunity 2004;5[5]:417-425.

  (9) von Aulock S, Schröder NWJ, Gueinzius K, Traub S, Hoffmann S, Graf K et al. Heterozygous toll-like receptor 4 polymorphism does not influence lipopolysaccharide-induced cytokine release in human whole blood. J Infect Dis 2003;188[6]:938-943.

  (10) Erridge C, Stewart J, Poxton IR. Monocytes Heterozygous for the Asp299Gly and Thr399Ile Mutations in the Toll-like Receptor 4 Gene Show No Deficit in Lipopolysaccharide Signalling. J Exp Med 2003;197[12]:1787-1791.

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  Conflict of Interest:
  None declared.

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• Published on: 18 April 2016

  Reply:

  • Denis Franchimont
  • Other Contributors:
    • Severine Vermeire, Jacques Deviere, and Paul Rutgeerts

  Dear Editor

  We would like to thank Doctor Ian Arnott for this very interesting comment.[1] Needless to say that we all agree that great caution should apply when reporting positive or negative association studies because of issues such as sample size, cryptic population substructure or phenotype misclassification. To this end, a TDT should always be performed to alleviate skepticism and doubts. It is true that a...

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  Dear Editor

  We would like to thank Doctor Ian Arnott for this very interesting comment.[1] Needless to say that we all agree that great caution should apply when reporting positive or negative association studies because of issues such as sample size, cryptic population substructure or phenotype misclassification. To this end, a TDT should always be performed to alleviate skepticism and doubts. It is true that a clear genetic heterogeneity emerges when examining NOD2 and TLR4 carriage frequencies in patients with Crohn’s disease in Europe, North America and Asia. That Crohn’s disease is a heterogeneous and polygenic disease is obvious in the light of recent genome wide screens. However, if multiple genes may contribute to Crohn’s disease, their relative impact may vary from one phenotype to another (or from one Crohn’s disease to another Crohn’s disease) but also, maybe, from one population to another (or from one ethnic background to another). In fact, the relative proportion of polymorphisms in susceptible genes that influence a specific phenotype may vary depending on the ethnic background. As pointed out by the Edinburgh group, the population attributable risk for NOD2 variants is significantly lower in Scotland and Ireland than in the rest of Europe or United States suggesting a much lower contribution of NOD2 variants (or perhaps of PRRs) in Crohn’s disease.[2] Interestingly enough, this is reinforced by their recent observation of a negative association of Crohn’s disease with the TLR4 Asp299Gly polymorphism. High-resolution haplotype mapping of the TLRs and signaling molecules should allow us to discriminate the relative influence of these PRRs in Crohn’s disease and UC across the world.

  References

  1. Arnott IDR, Ho G-T, Nimmo ER, Satsangi J. The toll like receptor-4 gene in IBD: further evidence for genetic heterogeneity in Europe [electronic response to Franchimont D, Vermeire S, El Housni H, Pierik M, Van Steen K, Gustot T, Quertinmont E, Abramowicz M, Van Gossum A, Devière J, and Rutgeerts P. Deficient host-bacteria interactions in inflammatory bowel disease? The toll-like receptor (TLR)-4 Asp299gly polymorphism is associated with Crohn’s disease and ulcerative colitis] gutjnl.com 2004 http://gut.bmjjournals.com/cgi/eletters/53/7/987#558.

  2. Arnott ID, Nimmo ER et al. NOD2/CARD15, TLR4 and CD14 mutations in Scottish and Irish Crohn's disease patients: evidence for genetic heterogeneity within Europe? Genes Immun 2004 Jun 10.

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  Conflict of Interest:
  None declared.

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• Published on: 18 April 2016

  The toll like receptor-4 gene in IBD: further evidence for genetic heterogeneity in Europe

  • Ian DR Arnott, Consultant Gastroentereologist
  • Other Contributors:
    • Gwo-Tzer Ho, Elaine R Nimmo, Jack Satsangi

  Dear Editor

  There is now strong evidence implicating the enteric flora in aeitiopathogenesis of IBD and the identification of CARD15 (NOD2) as a pattern recognition receptor (PRR) has given novel insights in to host bacteria interactions. CARD15 is implicated as the intracellular sensor of muramyl dipeptide, a highly conserved bacterial peptidoglycan motif, and raises the question whether other PRRs are involved...

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  Dear Editor

  There is now strong evidence implicating the enteric flora in aeitiopathogenesis of IBD and the identification of CARD15 (NOD2) as a pattern recognition receptor (PRR) has given novel insights in to host bacteria interactions. CARD15 is implicated as the intracellular sensor of muramyl dipeptide, a highly conserved bacterial peptidoglycan motif, and raises the question whether other PRRs are involved in the pathogenesis of Crohn’s disease (CD).

  Toll-like receptor 4 (TLR4), in combination with CD14, LBP and MD-2, acts as the PRR for the lipid-A moiety of lipopolysaccharride, a major component of gram-negative bacteria. Two common co-segregating polymorphisms of this gene have been described in humans, Asp299Gly and Thr399Ile. Asp299Gly has been associated with reduced bronchial responsiveness following LPS stimulation[1] although recent data have questioned the functional effect of this variant.[2]

  We therefore note with interest the article of Franchimont et al. reporting the Asp299Gly frequency in a Belgian population with IBD3.[3] Variant alleles were associated with both CD and ulcerative colitis (UC) in 2 cohorts and the allele was preferentially transmitted from carriers to affected subjects in a transmission disequilibrium test.

  However, apparently contradictory data from elsewhere in Europe highlight the difficulties of interpretation of genetic association studies from single populations. Torok et al examined the presence of both the Asp299Gly and Thr399Ile polymorphisms in a smaller German IBD population.[4] In contrast to the Belgian data, this group identified an association of Thr399Ile with UC but there was no association with Asp299Gly and no association with CD. In addition, we have previously published data on Asp299Gly in 480 Scottish patients with IBD and found no association with either CD or UC.[5]

  Why are these data sets discrepant? Issues relating to statistical power, population stratification in case-control studies, and phenotypic heterogeneity within inflammatory bowel disease may contribute, but we suggest more detailed examination of these data (Table 1) provides evidence for genetic heterogeneity between populations in Europe.

  CD genotype frequencies are very similar in the Leuven and Edinburgh data sets, and not significantly different from the German CD results. However, allelic frequencies in healthy controls are significantly different in the Scottish population, compared with European controls (8.8% v 4.6%, p=0.008, OR1.47, CI 1.2-3.5).

  It is clearly relevant that a strong suggestion of heterogeneity between populations is given in the original description of Arbour et al, where control population allelic frequencies for Asp299Gly ranged from 3.3 -7.9% in French and North American Data sets. It is noteworthy that these allelic variants are absent in the Japanese population.[6]

  Moreover, there is now compelling evidence for genetic heterogeneity between populations for CARD15 in both healthy controls and CD patients. Variant alleles are absent from Asian CD and control populations and exist at a lower frequency in African Americans CD patients and Ghanaian controls (carriage 1%).[7,8] Carriage frequencies in CD patients (approaching 50%) have been documented in Caucasian populations from North American and Central Europe. There is striking evidence of heterogeneity within Europe, and evidence for a geographical North-South gradient in gene effect, as for the CFTR delta 508 mutation in cystic fibrosis. Lower CARD15 (NOD2) frequencies in CD have been reported from Scotland [5] and Finland[9] and are absent in a small Icelandic population[10].

  These data illustrate further the real difficulties in candidate gene analysis in complex diseases. It is likely that the contribution of individual genetic determinants will differ between populations. We suggest that further genetic (as well as functional) data are required before the exact contribution of inherited variants of the TLR4 gene can be confirmed.

  Table 1 Number of patients studied and TLR4 Asp299Gly allelic frequencies in CD, UC and healthy control (HC) populations from Leuven, Munich and Edinburgh. HC allele frequencies differ between Edinburgh and Munich (p<_0.02 or="or" _1.35="_1.35" ci="ci" _1.1-4.5="_1.1-4.5" and="and" those="those" between="between" edinburgh="edinburgh" leuven="leuven" approach="approach" significance="significance" p="0.06)." a="a" comparison="comparison" of="of" the="the" cd="cd" genotype="genotype" frequencies="frequencies" is="is" also="also" shown="shown" for="for" _3="_3" studies.br="studies.br"/>  

  	Patient Numbers and Allele Frequencies (in brackets)			Crohn’s Disease Genotype Frequencies
  Place	CD	UC	HC	Wild type	Heterozygous	Homozygous
  Leuven	334 (11.0%)	163 (10.0%)	139 (5.0%)	79.3	19.5	1.2
  Munich	102 (7.0%)	98 (9.0%)	85.0	15.0	15.0	0
  Edinburgh	234 (10.3%)	246 (6.8%)	189 (8.8%)	79.5	19.7	0.8

  References

  (1) Arbour NC, Lorenz E, et al. TLR4 mutations are associated with endotoxin hyporesponsiveness in humans. Nat Genet. 2000 Jun;25(2):187-91.

  (2) Erridge C, Stewart J, Poxton IR. Monocytes heterozygous for the Asp299Gly and Thr399Ile mutations in the Toll-like receptor 4 gene show no deficit in lipopolysaccharide signalling.J Exp Med. 2003 Jun 16;197(12):1787-91.

  (3) Franchimont D, Vermeire S, et al. Deficient host-bacteria interactions in inflammatory bowel disease? The toll-like receptor (TLR)-4 Asp299gly polymorphism is associated with Crohn's disease and ulcerative colitis. Gut. 2004 Jul;53(7):987-92.

  (4) 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 Jul;112(1):85-91.

  (5) Arnott ID, Nimmo ER, et al. NOD2/CARD15, TLR4 and CD14 mutations in Scottish and Irish Crohn's disease patients: evidence for genetic heterogeneity within Europe? Genes Immun. 2004 Jun 10

  (6) Okayama N, Fujimura K, et al. 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;16:56-8.

  (7) Marsh S, McLeod HL. Crohn's disease: ethnic variation in CARD15 genotypes. Gut. 2003 May;52(5):770.

  (8) Bonen DK, Cho JH. The genetics of inflammatory bowel disease. Gastroenterology. 2003 Feb;124(2):521-36. Review.

  (9) Helio T, Halme L, et al. CARD15/NOD2 gene variants are associated with familially occurring and complicated forms of Crohn's disease. Gut. 2003 Apr;52(4):558-62.

  (10) Thjodleifsson B, Sigthorsson G, et al. Subclinical intestinal inflammation: an inherited abnormality in Crohn's disease relatives? Gastroenterology. 2003 Jun;124(7):1728

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  Conflict of Interest:
  None declared.

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