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The Endotoxin Receptor TLR4 Polymorphism Is Not Associated With Diabetes or Components of the Metabolic Syndrome

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 (14–17). 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

TOP RESEARCH DESIGN AND METHODS RESULTS AND DISCUSSION REFERENCES

 
Subjects.
The Cooperative Health Research in the Augsburg Region (KORA) Survey 2000 studied a population-based sample of 4,261 subjects aged 25–74 years during 1999–2001 (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 55–74 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 manufacturer’s 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

TOP RESEARCH DESIGN AND METHODS RESULTS AND DISCUSSION REFERENCES

 
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.

	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

TOP RESEARCH DESIGN AND METHODS RESULTS AND DISCUSSION REFERENCES

 

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