Correspondence

Toll-like Receptor 4 Polymorphisms and Atherogenesis

N Engl J Med 2002; 347:1978-1980December 12, 2002

Article

To the Editor:

Kiechl and colleagues (July 18 issue)1 provide interesting evidence that the toll-like receptor 4 (TLR4) 896G (299Gly) polymorphism may confer susceptibility to bacterial infection. As part of ongoing collaborative projects,2-4 we have investigated the role of this polymorphism in a large United Kingdom study of invasive pneumococcal disease and a large West African study of pulmonary tuberculosis. There was no evidence of an association between the TLR4 896G allele and invasive pneumococcal disease or pulmonary tuberculosis (Table 1Table 1Association between the Toll-like Receptor 4 896G Allele and Invasive Pneumococcal Disease or Pulmonary Tuberculosis.). These data add to the findings of Read et al.5 that the TLR4 896G polymorphism was not associated with susceptibility to, or severity of, meningococcal disease. Together, they suggest that this polymorphism is likely to influence the course of infection only by specific organisms, not by a broad spectrum.

The study by Kiechl et al. included eight persons with putative bacterial infection who carried the TLR4 896G allele. It would be informative to know which infectious pathogens were identified in the study participants and how the authors' analysis might be affected by excluding those without confirmed bacterial infection. Exclusion of particular pathogens (for example, Streptococcus pneumoniae, Mycobacterium tuberculosis, and Neisseria meningitidis) might allow a clearer identification of significant biologic effects. The effects of the TLR4 polymorphism that have been identified to date have been small at best, and confirmation of an association with infectious disease will require large, well-designed studies with precise clinical and microbiologic phenotyping.

Graham S. Cooke, M.R.C.P.
Shelley Segal, M.R.C.P.
Adrian V.S. Hill, M.D., D.Phil.
Wellcome Trust Centre for Human Genetics, Oxford OX3 7BN, United Kingdom

for the Tuberculosis, Genetics, and Environment (TBGENENV) and the Oxford Pneumococcal Surveillance Study Groups

5 References

1. 1

   Kiechl S, Reindl M, Wiedermann CJ, et al. Toll-like receptor 4 polymorphisms and atherogenesis. N Engl J Med 2002;347:185-192
   Full Text | Web of Science | Medline

2. 2

   Bellamy R, Ruwende C, Corrah T, McAdam KPWJ, Whittle HC, Hill AVS. Variations in the NRAMP1 gene and susceptibility to tuberculosis in West Africans. N Engl J Med 1998;338:640-644
   Full Text | Web of Science | Medline

3. 3

   Bennett S, Lienhardt C, Bah-Sow O, et al. Investigation of environmental and host-related risk factors for tuberculosis in Africa. II. Investigation of host genetic factors. Am J Epidemiol 2002;155:1074-1079
   CrossRef | Web of Science | Medline

4. 4

   Roy S, Knox K, Segal S, et al. MBL genotype and risk of invasive pneumococcal disease: a case-control study. Lancet 2002;359:1569-1573
   CrossRef | Web of Science | Medline

5. 5

   Read RC, Pullin J, Gregory S, et al. A functional polymorphism of toll-like receptor 4 is not associated with likelihood or severity of meningococcal disease. J Infect Dis 2001;184:640-642
   CrossRef | Web of Science | Medline

To the Editor:

If “snake eyes” are thrown three times in a row (P=0.000021), can we conclude that the dice are loaded? Not if the dice have been rolled 3000 times, and triple snake eyes observed only once (P=0.062). The same principle makes us reluctant to accept the conclusion that the common white variant of TLR4 provides protection against atherosclerosis, as Kiechl and colleagues claim. They had many end points from which to choose — for example, femoral atherosclerosis was measured in the study population1 but was not reported as a comparison in the article.

In a population studied previously,2 71 patients had undergone carotid endarterectomy. In contrast to the findings of Kiechl et al., the number of patients who had undergone endarterectomy and who were carrying the mutant TLR4 genotype was actually larger than the number of carriers in a set of matched controls (Table 1Table 1TLR4 Promoter Genotypes of 70 Patients Who Underwent Carotid Endarterectomy and 68 Matched Controls.).

Even if statistically valid, a correlation may merely represent linkage with a nearby mutation that actually confers a risk, or it may be due to population structure; the prevalence of the compound TLR4 variant differs greatly between populations.3 Small differences in the frequency of the TLR4 allele in phenotypically distinguishable groups may merely reflect residual inhomogeneity of the population that is being studied.

TLR4 evolved to detect lipopolysaccharide, a function that led to positional cloning of the gene in 1998.4 Strong circumspection is warranted with regard to other functions. Genotyping is easily achieved in large populations and may disclose risk factors for disease, but exceptional caution must be exercised when interpreting the data.

Bruce Beutler, M.D.
Ernest Beutler, M.D.
Scripps Research Institute, La Jolla, CA 92037
bruce@scripps.edu

4 References

1. 1

   Mayr M, Kiechl S, Willeit J, Wick G, Xu Q. Infections, immunity, and atherosclerosis: associations of antibodies to Chlamydia pneumoniae, Helicobacter pylori, and cytomegalovirus with immune reactions to heat-shock protein 60 and carotid or femoral atherosclerosis. Circulation 2000;102:833-839
   Web of Science | Medline

2. 2

   Beutler E, Felitti VJ, Koziol JA, Ho NJ, Gelbart T. Penetrance of 845G→A (C282Y) HFE hereditary haemochromatosis mutation in the USA. Lancet 2002;359:211-218
   CrossRef | Web of Science | Medline

3. 3

   Smirnova I, Hamblin MT, McBride C, Beutler B, Di Rienzo A. Excess of rare amino acid polymorphisms in the Toll-like receptor 4 in humans. Genetics 2001;158:1657-1664
   Web of Science | Medline

4. 4

   Poltorak A, He X, Smirnova I, et al. Defective LPS signaling in C3H/HeJ and C57BL/10ScCr mice: mutations in Tlr4 gene. Science 1998;282:2085-2088
   CrossRef | Web of Science | Medline

Author/Editor Response

The authors reply:

To the Editor: Cooke et al. report that the Asp299Gly TLR4 polymorphism is not associated with an increased risk of invasive pneumococcal infection, pulmonary tuberculosis, or meningococcal disease. The latter two types of infection are comparatively rare in Western countries and contribute little to the overall burden of severe acute infections. Although it is beyond doubt that TLR4 is not directly involved in the immune response to all pathogens, its relevancy to a broad spectrum of widespread and clinically relevant microorganisms (chlamydia, helicobacter, haemophilus, Escherichia coli, klebsiella, salmonella, porphyromonas, anaerobic pathogens, respiratory syncytial virus, candida, and many more) has been proposed. In our study, microbiologic phenotyping was not feasible for all infections for reasons such as pretreatment with antibiotics or a lack of cultures. This, however, does not diminish our finding of a significant association between the TLR4 polymorphism and the overall burden of severe acute infections in the general community.

Beutler and Beutler believe that we have selectively presented end points related to the TLR4 polymorphism and have omitted those without a significant association. We are surprised by such a statement, because our analyses followed a uniform protocol, and all main longitudinal end points were carefully considered. In fact, we observed a similar protective effect of the Asp299Gly TLR4 polymorphism in the development of femoral-artery atherosclerosis (Table 1Table 1Association between the Asp299Gly Toll-like Receptor 4 (TLR4) Polymorphism and Incident Femoral-Artery Atherosclerosis (1995–2000) in the Bruneck Study.).

Beutler and Beutler further state that correlations may merely reflect a linkage with nearby mutations. This possibility cannot be ruled out, as we discussed in our article, but it seems unlikely, because the functional significance of the TLR4 polymorphism is well documented in vivo and in cellular transfection studies,1 and the experimental and epidemiologic findings are highly consistent.

Finally, Beutler and Beutler present data from a small case–control study comparing patients who underwent carotid endarterectomy with controls. Even if we assume that this study is free from bias, that it is adequately powered, and that our Asp299Gly and their TLR4 promoter polymorphisms are functionally equivalent, the findings are not in conflict with ours, because they tested the wrong hypothesis: pathogenic mechanisms underlying vessel stenosis are substantially different from those involved in atherogenesis. We have previously shown that the development of carotid stenosis relies primarily on atherothrombosis and on procoagulant risk factors2,3 and that this advanced stage in vessel disease is not significantly related to inflammation and infectious illness.4

Stefan Kiechl, M.D.
Johann Willeit, M.D.
University of Innsbruck, A-6020 Innsbruck, Austria

David A. Schwartz, M.D.
Duke University Medical Center, Durham, NC 17710

4 References

1. 1

   Arbour NC, Lorenz E, Schutte BC, et al. TLR4 mutations are associated with endotoxin hyporesponsiveness in humans. Nat Genet 2000;25:187-191
   CrossRef | Web of Science | Medline

2. 2

   Kiechl S, Willeit J. The natural course of atherosclerosis. I. Incidence and progression. Arterioscler Thromb Vasc Biol 1999;19:1484-1490
   CrossRef | Web of Science | Medline

3. 3

   Willeit J, Kiechl S, Oberhollenzer F, et al. Distinct risk profiles of early and advanced atherosclerosis: prospective results from the Bruneck Study. Arterioscler Thromb Vasc Biol 2000;20:529-537
   CrossRef | Web of Science | Medline

4. 4

   Kiechl S, Egger G, Mayr M, et al. Chronic infections and the risk of carotid atherosclerosis: prospective results from a large population study. Circulation 2001;103:1064-1070
   Web of Science | Medline

Citing Articles (17)

Citing Articles

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   Mamoona Noreen, Muhammad Ali A. Shah, Sheeba Murad Mall, Shazia Choudhary, Tahir Hussain, Iltaf Ahmed, Syed Fazal Jalil, Muhammad Imran Raza. (2012) TLR4 polymorphisms and disease susceptibility. Inflammation Research
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   T E West, W Chierakul, N Chantratita, D Limmathurotsakul, V Wuthiekanun, M J Emond, T R Hawn, S J Peacock, S J Skerrett. (2011) Toll-like receptor 4 region genetic variants are associated with susceptibility to melioidosis. Genes and Immunity
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   Hanna Suhir, Amos Etzioni. (2010) The Role of Toll-Like Receptor Signaling in Human Immunodeficiencies. Clinical Reviews in Allergy & Immunology 38:1, 11-19
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   S. F. Martin, J. C. Dudda, E. Bachtanian, A. Lembo, S. Liller, C. Durr, M. M. Heimesaat, S. Bereswill, G. Fejer, R. Vassileva, T. Jakob, N. Freudenberg, C. C. Termeer, C. Johner, C. Galanos, M. A. Freudenberg. (2008) Toll-like receptor and IL-12 signaling control susceptibility to contact hypersensitivity. Journal of Experimental Medicine 205:9, 2151-2162
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   April Kaur Randhawa, Thomas R Hawn. (2008) Toll-like receptors: their roles in bacterial recognition and respiratory infections. Expert Review of Anti-infective Therapy 6:4, 479-495
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6. 6

   M. Veltkamp, J. C. Grutters, C. H. M. van Moorsel, H. J. T. Ruven, J. M. M. van den Bosch. (2006) Toll-like receptor (TLR) 4 polymorphism Asp299Gly is not associated with disease course in Dutch sarcoidosis patients. Clinical and Experimental Immunology 145:2, 215-218
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   Dianhua Jiang, Jiurong Liang, Yuhang Li, Paul W Noble. (2006) The role of Toll-like receptors in non-infectious lung injury. Cell Research 16:8, 693-701
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   Tryfon Vainas, Frank R.M. Stassen, Cathrien A. Bruggeman, Rob J. Th. J. Welten, Luc H.J.M. van den Akker, Peter J.E.H.M. Kitslaar, Amado S. Peña, Servaas A. Morré. (2006) Synergistic effect of Toll-like receptor 4 and CD14 polymorphisms on the total atherosclerosis burden in patients with peripheral arterial disease. Journal of Vascular Surgery 44:2, 326-332
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9. 9

   Stuart E. Turvey, Thomas R. Hawn. (2006) Towards subtlety: Understanding the role of Toll-like receptor signaling in susceptibility to human infections. Clinical Immunology 120:1, 1-9
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10. 10

    Kazunori Shimada, Hiroyuki Daida, Wei Ma-Krupa, Jörg J Goronzy, Cornelia M Weyand. (2005) Lipopolysaccharide, CD14 and Toll-like receptors: an emerging link between innate immunity and atherosclerotic disease. Future Cardiology 1:5, 657-674
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11. 11

    G. D. NORATA, K. GARLASCHELLI, M. ONGARI, S. RASELLI, L. GRIGORE, F. BENVENUTO, F. M. MAGGI, A. L. CATAPANO. (2005) Effect of the Toll-like receptor 4 (TLR-4) variants on intima-media thickness and monocyte-derived macrophage response to LPS. Journal of Internal Medicine 258:1, 21-27
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12. 12

    Yi-Chun Lin, Ying-Ming Chang, Jia-Ming Yu, Jeng-Hsien Yen, Jan-Gowth Chang, Chaur-Jong Hu. (2005) Toll-like receptor 4 gene C119A but not Asp299Gly polymorphism is associated with ischemic stroke among ethnic Chinese in Taiwan. Atherosclerosis 180:2, 305-309
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13. 13

    Markus Mueller, Stefan Postius, Jean G. Thimm, Katja Gueinzius, Inge Muehldorfer, Corinna Hermann. (2004) Toll-like receptors 2 and 4 do not contribute to clearance of Chlamydophila pneumoniae in mice, but are necessary for the release of monokines. Immunobiology 209:8, 599-608
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14. 14

    Aryan Vink, Dominique PV de Kleijn, Gerard Pasterkamp. (2004) Functional role for toll-like receptors in atherosclerosis and arterial remodeling. Current Opinion in Lipidology 15:5, 515-521
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15. 15

    Lutz Hamann, Axel Hamprecht, Abuzeid Gomma, Ralf R Schumann. (2004) Rapid and inexpensive real-time PCR for genotyping functional polymorphisms within the Toll-like receptor -2, -4, and -9 genes. Journal of Immunological Methods 285:2, 281-291
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16. 16

    I. A. Yang, S. T. Holgate, J. W. Holloway. (2004) Toll-like receptor polymorphisms and allergic disease: interpreting the evidence from genetic studies. Clinical <html_ent glyph="@amp;" ascii="&"/> Experimental Allergy 34:2, 163-166
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17. 17

    Cécile M.C Fremond, Delphine M.M Nicolle, David S Torres, Valérie F.J Quesniaux. (2003) Control of Mycobacterium bovis BCG infection with increased inflammation in TLR4-deficient mice. Microbes and Infection 5:12, 1070-1081
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