Asp299Gly and Thr399Ile Genotypes of the TLR4 Gene Are Associated With a Reduced Prevalence of Diabetic Neuropathy in Patients With Type 2 Diabetes GOTTFRIED RUDOFSKY,JR., MD 1 PETER REISMANN 1 STEFFEN WITTE, BOS 2 PER M. HUMPERT, MD 1 BEREND ISERMANN, MD 1 TRIANTAFYLLOS CHAVAKIS, MD 1 J¨ ORG TAFEL, MD 1 VALERY V. NOSIKOV, PHD 3 ANDREAS HAMANN, MD 1 PETER NAWROTH, MD 1 ANGELIKA BIERHAUS, PHD 1 OBJECTIVE — To establish whether single nucleotide polymorphisms (Asp299Gly and Thr399Ile) of the toll-like receptor 4 have an association with late diabetic complications. RESEARCH DESIGN AND METHODS — The study was conducted in 246 type 1 and 530 type 2 diabetic patients. The alleles of both polymorphisms were detected using PCR and subsequent cleavage by NcoI and HinfI restriction endonucleases. RESULTS — No difference was found between type 1 and type 2 diabetic patients in the prevalence of alleles of the Asp299Gly and Thr399Ile polymorphisms. In most cases, the alleles Gly299 and Ile399 occurred in a co-segregatory manner. The prevalence of the Gly299/Ile399 haplotype was 10.6 and 12.1% in type 1 and type 2 diabetic patients, respectively (P = 0.63). No association with diabetic nephropathy or diabetic neuropathy was found in type 1 diabetic patients. In type 2 diabetic patients, however, heterozygote carriers of the Asp299Gly and Thr399Ile genotypes had a significantly reduced prevalence of diabetic neuropathy (odds ratio 0.35 [95% CI 0.19 – 0.61]; P = 0.0002); no association with diabetic nephropathy was found. CONCLUSIONS — Our data indicate that Asp299Gly and Thr399Ile genotypes of the TLR4 gene are associated with reduced prevalence of diabetic neuropathy in type 2, but not in type 1, diabetes. Thus different mechanisms may be involved in the pathophysiology of diabetic neu- ropathy in type 1 and type 2 diabetes. Diabetes Care 27:179 –183, 2004 R ecently, our understanding of type 2 diabetes has changed consider- ably. Levels of C-reactive proteins have been shown to predict the occur- rence of type 2 diabetes. Studies in animal models as well as humans have suggested that type 2 diabetes might be associated with changes in the innate immune re- sponse (1–5). Furthermore, experiments in which the mitogen-activated protein kinase or inhibitor B-kinase pathways are genetically controlled have shown that activator protein-1 and nuclear factor (NF)-B are central regulators not only of inflammatory reactions (6), but also of the insulin response and glucose metabolism (7,8). In addition, one of the receptors important for developing late diabetic complications, the receptor for advanced glycation end products (RAGE), has been shown to participate in the innate im- mune response and behave as a pattern recognition receptor (9,10). This implies that factors regulating the innate immune response might be also involved in late diabetic complications. One of the central regulators of the innate immune response is the toll-like receptor (TLR)-4 (11). The recognition of microbial components by mammalian TLRs plays an important role in activation of the innate immune re- sponse and subsequent proinflammatory reactions. In addition to binding lipo- polysaccharide (LPS), TLR-4 also inter- acts with endogenous ligands such as oxLDL, heat shock proteins 60 and 70, fibrinogen, and fibronectin (11,12), which are also elevated in diabetes (13– 17). Two common single nucleotide poly- morphisms have been found in the cod- ing region of the human TLR4 gene at exon 3 that are in a tight linkage disequi- librium (18). They lead to amino acid ex- changes in positions 299 (Asp299Gly) and 399 (Thr399Ile). It has been shown that the presence of Gly299 and Ile399 alters the structure of the extracellular domain of TLR-4 (19), which might influence the ligand binding. The functional signifi- cance of these polymorphisms has re- cently been demonstrated in experiments showing an association of hyporespon- siveness to inhaled LPS (19). The carriers of the Asp299Gly genotype have reduced ●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●● From the 1 Department Medicine I, University of Heidelberg, Heidelberg, Germany; the 2 Department of Medical Biometry, University of Heidelberg, Heidelberg, Germany; and the 3 Department of Molecular Diagnostics and Genomic Fingerprinting, National Research Centre “GosNII Genetika,” Moscow, Russia. Address correspondence and reprint requests to Angelika Bierhaus, PhD, Medizinische Klinik I der Universita ¨ t Heidelberg, Otto-Meyerhof-Zentrum, Im Neuenheimer Feld 350, 69120 Heidelberg, Germany. E-mail: angelika_bierhaus@med.uni-heidelberg.de. Received for publication 23 April 2003 and accepted in revised form 5 October 2003. G.R. and P.R. contributed equally to this study. Abbreviations: AF, allelic frequency; CR, carrier rate; LPS, lipopolysaccharide; NF, nuclear factor; RAGE, receptor for advanced glycation end products; TLR, toll-like receptor. A table elsewhere in this issue shows conventional and Syste `me International (SI) units and conversion factors for many substances. © 2004 by the American Diabetes Association. Pathophysiology/Complications O R I G I N A L A R T I C L E DIABETES CARE, VOLUME 27, NUMBER 1, JANUARY 2004 179