The Endotoxin Receptor TLR4 Polymorphism Is Not Associated With Diabetes or Components of the Metabolic Syndrome
Thomas Illig1,
Friedhelm Bongardt1,
Andreas Schöpfer1,
Rolf Holle2,
Sylvia Müller3,
Wolfgang Rathmann4,
Wolfgang Koenig5,
Christa Meisinger1,
H.-Erich Wichmann1, and
Hubert Kolb3 For The KORA Study Group1,2
1 GSF National Research Center for Environment and Health, Institute of Epidemiology, Neuherberg, Germany
2 GSF National Research Center for Environment and Health, Institute of Health Economics and Health Care Management, Neuherberg, Germany
3 German Diabetes Clinic, German Diabetes Research Institute at the University of Düsseldorf, Düsseldorf, Germany
4 Department of Biometrics and Epidemiology, German Diabetes Research Institute at the University of Düsseldorf, Düsseldorf, Germany
5 Department of Internal Medicine II-Cardiology, Medical Center, University of Ulm, Ulm, Germany
The gene coding for the endotoxin receptor TLR4 (toll-like receptor 4) has been sequenced recently, and the polymorphic spectrum of the gene has been elucidated (1). A polymorphism in the gene affects the inflammatory response to lipopolysaccharide (LPS) (2,3), with impact on the risk of gram-negative infections and septic shock (4,5). We have analyzed two cosegregating mutations in the gene region coding for the extracellular domain of the endotoxin receptor TLR4, characterized by a substitution at amino acid position 299 (glycine for aspartate) and another at position 399 (isoleucine for threonine). The 299Gly form of TLR4 affects the inflammatory response to LPS by exhibiting attenuated signaling leading to a dampened response to LPS (4,5). Polymorphisms in the TLR4 protein show an increased risk to develop a septic shock with gram-negative microorganisms (6), premature birth (7), and graft versus host disease after hematopoietic stem cell transplantation (8).
Polymorphisms in the TLR4 gene, however, did not reveal any association with severity of menigococcal disease (9), risk of premature rupture of membranes caused by infections (10), or asthma- and atopy-related diseases (11).
TLR4 interacts with endogenous ligands such as the stress protein hsp60 (12). Since hsp60 is an important player in chronic inflammatory conditions (12), the TLR4 polymorphism may regulate the subclinical inflammation underlying the pathogenesis of arteriosclerosis.
Indeed, a recent study reported an association of the TLR4 polymorphism with atherogenesis (13). Subjects carrying the rare 299Gly allele exhibited a lower risk of carotid atherosclerosis and a smaller intima-media thickness in the common carotid artery (13).
Parameters of mild systemic inflammation observed in association with atherosclerosis are strikingly similar to what is seen in subjects with metabolic syndrome or type 2 diabetes, notably elevated serum levels of acute-phase proteins, some inflammatory cytokines, and soluble adhesion molecules (1417). We therefore analyzed for an association of the TLR4 gene polymorphism with features of the metabolic syndrome or with overt type 2 diabetes.
 |
RESEARCH DESIGN AND METHODS
|
---|
Subjects.
The Cooperative Health Research in the Augsburg Region (KORA) Survey 2000 studied a population-based sample of 4,261 subjects aged 2574 years during 19992001 (14). Each study participant signed a consent form to participate in genetic studies. All study methods were approved by the ethics committee of the "Bayerische Landesärtzekammer" Munich. The sampling design followed the guidelines of three previous surveys in the same region as part of the multinational World Health Organization (WHO)-MONICA (Monitoring Trends and Determinants of Cardiovascular Disease) study. In the age range 5574 years, 1,653 people participated in a standardized interview followed by biochemical and clinical analyses. An oral glucose tolerance test and biochemical and immunological analyses were performed as described previously (18). Acute infections (fever) or gastrointestinal illness were an exclusion criterion for the oral glucose tolerance test. Diabetes was diagnosed according to 1999 WHO criteria (18). After exclusion of all subjects with self-reported type 1 diabetes, humoral autoimmunity to glutamic acid decarboxylase, or diabetes onset in the context of pancreatitis, a total of 236 individuals with type 2 diabetes and 242 individuals with impaired glucose tolerance (IGT) were available for analyses. There were 244 normoglycemic control subjects randomly selected after matching for age and sex. This totals 722 probands. Of the diabetic patients, 120 were newly detected and did not yet receive antidiabetic treatment, of the other 116, 33% were under insulin treatment; 57% took oral antidiabetic agents (18).
Biochemical analyses.
Concentrations of C-reactive protein (CRP), SAA (serum amyloid A), and fibrinogen in plasma were determined by nephelometric assays as described (14,19,20). Serum concentrations of cytokines and circulating receptors were determined by rigidly evaluated sandwich enzyme-linked immunosorbent assay; lipid diagnostics were done by standard procedures (14).
Genotyping.
Genomic DNA was extracted from leukocytes by using the Puregene DNA Isolation Kit (Gentra Systems, Minneapolis, MN) according to the manufacturers recommendation. For polymorphism analysis DNA was subjected to PCR and homogenous MassEXTEND reaction (hME; Sequenom, San Diego, CA). The identical SNPs as described were analyzed (4).
Statistical analysis.
The nonparametrical Kruskal-Wallis test was performed to test for association with characteristics of subclinical inflammation. All other variables were analyzed with ANOVA for continuous end points and logistic regression for discrete end points. All models were adjusted for age and sex.
 |
RESULTS AND DISCUSSION
|
---|
As observed by Kiechl et al. (13), the Asp299Gly and Thr399Ile polymorphisms were in linkage disequilibrium in >90% of cases. Therefore, we report the analyses with regard to the Asp299Gly polymorphism only. Results for the polymorphism at amino acid 399 provided were not different. However, the functional defect of TLR4 is largely caused by the amino acid substitution at position 299.
Genotype analysis of the polymorphism resulting in an Asp299Gly exchange was possible in 671 of the 722 subjects leading to a call rate of 92.9%. The Asp299/Asp299 was observed in 599 cases (89.3%), the heterozygous type Asp299/299Gly was found in 70 cases (10.4%), and the homozygous type 299Gly/299Gly in 2 cases (0.3%). A bias due to the genotyping rate of 92.9% appears unlikely because both single nucleotide polymorphisms were in Hardy Weinberg equilibrium and the data are very similar to previously published data (13).
The group with type 2 diabetes or IGT exhibited characteristics of subclinical inflammation with elevated systemic levels of IL-6, IL-6 receptor, CRP, SAA, or fibrinogen (Table 1). This higher state of activation of innate immunity was not accompanied by a bias toward the high LPS responder type (TLR4 major allele, Table 1). In this context it should be noted that individuals taking anti-inflammatory drugs were not excluded because, in the age-group studied, a large fraction were taking such substances, including statins, antihypertensive medication, and antidiabetic drugs. The impact of such therapy appears limited because systemic levels of inflammatory mediators are elevated in individuals with type 2 diabetes, metabolic syndrome, or atherosclerosis (14) despite their more frequent use of such drugs.
Next, we analyzed for an association of the TLR4 polymorphism with individual parameters of the metabolic syndrome or of subclinical inflammation. Subjects with one or two alleles causing the 299Gly TLR4 did not differ from carriers of TLR4 Asp homozygotes with regard to hypertension, BMI, waist circumference, or HDL cholesterol levels (Table 2). Differences were also not observed for systemic levels of IL-6, IL-6 receptor, CRP, SAA, or fibrinogen (Table 2). Of all parameters analyzed, only the prevalence of hypertension showed a trend (P = 0.07), leaving the possibility of a mild protective effect of the Gly299 TLR4 allele. A similar analysis only regarding control and IGT subjects also failed to show an association between TLR4 genotype and metabolic or immunologic phenotype (Table 3).
View this table:
[in this window]
[in a new window]
|
TABLE 2 No association of TLR4 polymorphism with components of the metabolic syndrome or of subclinical systemic inflammation
|
|
View this table:
[in this window]
[in a new window]
|
TABLE 3 Analysis of the influence of the TLR4 genotype on components of the metabolic syndrome or of subclinical systemic inflammation, regarding IGT subjects and control subjects solely
|
|
The data do not confirm TLR4 allele-dependent differences in systemic IL-6 or fibrinogen concentrations reported for the Bruneck Study (13). Both the Bruneck and our study share basic characteristics of population-based sampling and a similar age range (4080 vs. 5574 years) and number of subjects studied. Methodological aspects probably do not account for the difference because a number of inflammatory markers were assessed in both studies. None of these came close to the significance level in our study, while most parameters did so in the Bruneck Study. A reason for this difference could not be identified; in theory small differences may be expected with regard to the genetic background, environmental factors (such as low level infections), or the sampling procedure. In this context it is noteworthy that almost all carriers of TLR4 299Gly allele are heterozygous and hence coexpress the fully functional TLR4 Asp299. Monocytes heterozygous for TLR4 do not exhibit deficient responses to LPS (3,21). Independent of this issue, the important finding of our KORA study is that there is no strong impact of the TLR4 polymorphism on major features of the metabolic syndrome.
 |
ACKNOWLEDGMENTS
|
---|
This work was supported by grants from the German National Genome Research Net (NGFN) platform 6 and by the GSF Research Center, the Deutsche Forschungsgemeinschaft, the European Foundation for the Study of Diabetes, the Federal Ministry of Health, the Ministry of Science and Research of North Rhine-Westfalia, and the Department of Internal Medicine II-Cardiology at the University of Ulm.
This study was supported by the KORA study group: A. Döring, T. Illig, H. Löwel, C. Meisinger, B. Thorand, and H.E.-Wichmann from the GSF National Research Center for Environment and Health, Institute of Epidemiology; R. Holle and J. John from the GSF National Research Center for Environment and Health, Institute of Health Economics and Health Care Management.
We thank Monika Wimmer, Michaela Bunge, and Petra Weskamp for excellent technical assistance. The genetic part of the work was performed in the "Genome Analysis Center" of the GSF Research Center for Environment and Health.
Address correspondence and reprint requests to Dr. Thomas Illig, GSF Research Center for Environment and Health, Building 34, Room 307a, Ingolstaedter Landstr. 1, D-85764 Munich-Neuherberg, Germany. E-mail: illig{at}gsf.de
Received for publication May 7, 2003
and accepted in revised form August 12, 2003
Key Words: CRP, C-reactive protein IGT, impaired glucose tolerance KORA, Cooperative Health Research in the Augsburg Region LPS, lipopolysaccharide SAA, serum amyloid A WHO, World Health Organization
 |
REFERENCES
|
---|
- Smirnova I, Poltorak A, Chan EK, McBride C, Beutler B: Phylogenetic variation and polymorphism at the toll-like receptor 4 locus (TLR4).
Genome Biol1
:RESEARCH002
, Epub2000[Medline]
- Schwartz DA: TLR4 and LPS hyporesponsiveness in humans.
Int J Hyg Environ Health205
:221
227,2002[Medline]
- Schmitt C, Humeny A, Becker CM, Brune K, Pahl A: Polymorphisms of TLR4: rapid genotyping and reduced response to lipopolysaccharide of TLR4 mutant alleles.
Clin Chem48
:1661
1667,2002[Abstract/Free Full Text]
- Arbour NC, Lorenz E, Schutte BC, Zabner J, Kline JN, Jones M, Frees K, Watt JL, Schwartz DA: TLR4 mutations are associated with endotoxin hyporesponsiveness in humans.
Nat Genet25
:187
191,2000[Medline]
- Agnese DM, Calvano JE, Hahm SJ, Coyle SM, Corbett SA, Calvano SE, Lowry SF: Human toll-like receptor 4 mutations but not CD14 polymorphisms are associated with an increased risk of gram-negative infections.
J Infect Dis186
:1522
1525,2002[Medline]
- Lorenz E, Mira JP, Frees KL, Schwartz DA: Relevance of mutations in the TLR4 receptor in patients with gram-negative septic shock.
Arch Intern Med162
:1028
1032,2002[Abstract/Free Full Text]
- Lorenz E, Hallman M, Marttila R, Haataja R, Schwartz DA: Association between the Asp299Gly polymorphisms in the toll-like receptor 4 and premature births in the Finnish population.
Pediatr Res52
:373
376,2002[Abstract/Free Full Text]
- Lorenz E, Schwartz DA, Martin PJ, Gooley T, Lin MT, Chien JW, Hansen JA, Clark JG: Association of TLR4 mutations and the risk for acute GVHD after HLA-matched-sibling hematopoietic stem cell transplantation.
Biol Blood Marrow Transplant7
:384
387,2001[Medline]
- Read RC, Pullin J, Gregory S, Borrow R, Kaczmarski EB, di Giovine FS, Dower SK, Cannings C, Wilson AG: A functional polymorphism of toll-like receptor 4 is not associated with likelihood or severity of meningococcal disease.
J Infect Dis184
:640
642,2001[Medline]
- Ferrand PE, Fujimoto T, Chennathukuzhi V, Parry S, Macones GA, Sammel M, Kuivaniemi H, Romero R, Strauss JF III: The CARD15 2936insC mutation and TLR4 896 A>G polymorphism in African Americans and risk of preterm premature rupture of membranes (PPROM).
Mol Hum Reprod8
:1031
1034,2002[Abstract/Free Full Text]
- Raby BA, Klimecki WT, Laprise C, Renaud Y, Faith J, Lemire M, Greenwood C, Weiland KM, Lange C, Palmer LJ, Lazarus R, Vercelli D, Kwiatkowski DJ, Silverman EK, Martinez FD, Hudson TJ, Weiss ST: Polymorphisms in toll-like receptor 4 are not associated with asthma or atopy-related phenotypes.
Am J Respir Crit Care Med166
:1449
1456,2002[Abstract/Free Full Text]
- Ohashi K, Burkart V, Flohe S, Kolb H: Cutting edge: heat shock protein 60 is a putative endogenous ligand of the toll-like receptor-4 complex.
J Immunol164
:558
561,2000[Abstract/Free Full Text]
- Kiechl S, Lorenz E, Reindl M, Wiedermann CJ, Oberhollenzer F, Bonora E, Willeit J, Schwartz DA: Toll-like receptor 4 polymorphisms and atherogenesis.
N Engl J Med347
:185
192,2002[Abstract/Free Full Text]
- Muller S, Martin S, Koenig W, Hanifi-Moghaddam P, Rathmann W, Haastert B, Giani G, Illig T, Thorand B, Kolb H: Impaired glucose tolerance is associated with increased serum concentrations of interleukin 6 and co-regulated acute-phase proteins but not TNF-alpha or its receptors.
Diabetologia45
:805
812,2002[Medline]
- Pickup JC, Crook MA: Is type II diabetes mellitus a disease of the innate immune system?
Diabetologia41
:1241
1248,1998[Medline]
- Festa A, DAgostino R, Howard G, Mykkanen L, Tracy RP, Haffner SM: Inflammation and microalbuminuria in nondiabetic and type 2 diabetic subjects: the Insulin Resistance Atherosclerosis study.
Kidney Int58
:1703
1710,2000[Medline]
- Greenberg AS, McDaniel ML: Identifying the links between obesity, insulin resistance and beta-cell function: potential role of adipocyte-derived cytokines in the pathogenesis of type 2 diabetes.
Eur J Clin Invest(32 Suppl. 3)
:24
34,2002
- Rathmann W, Haastert B, Icks A, Lowel H, Meisinger C, Holle R, Giani G: High prevalence of undiagnosed diabetes mellitus in Southern Germany: Target populations for efficient screening: the KORA survey 2000.
Diabetologia46
:182
189,2003[Medline]
- Rifai N, Tracy RP, Ridker PM: Clinical efficacy of an automated high-sensitivity C-reactive protein assay.
Clin Chem45
:2136
2141,1999[Abstract/Free Full Text]
- Hoffmeister A, Rothenbacher D, Bazner U, Frohlich M, Brenner H, Hombach V, Koenig W: Role of novel markers of inflammation in patients with stable coronary heart disease.
Am J Cardiol87
:262
266,2001[Medline]
- 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 Med197
:1787
1791,2003[Abstract/Free Full Text]