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J Immunol Methods. 2004 Jun;289(1-2):81-7.

Single step high-throughput determination of Toll-like receptor 4 polymorphisms.

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Research Laboratory of the Department of Clinical Chemistry, University Medical Center Utrecht, The Netherlands.



Toll-like receptors are central components of host defence in humans, responsible for recognition of pathogen-associated molecular patterns and activation of innate immunity. Toll-like receptor 4 (TLR4) is activated by lipopolysaccharide (LPS) and other microbial components, thereby initiating the expression and release of pro-inflammatory cytokines. The common, frequently co-segregating allelic variants Asp299Gly and Thr399Ile have been related to susceptibility to gram-negative infections and sepsis and may be involved in the development of atherosclerosis. Identification of TLR4 Asp299Gly and Thr399Ile genotypes can be important for examination of genotype/phenotype relationships as well as for individual risk assessment of patients.


TLR4 Asp299Gly and Thr399Ile genotypes were detected by a single tube polymerase chain reaction (PCR), based on exonuclease degradation of dual labelled allele-specific oligonucleotides. The assay results were compared with conventional restriction fragment length polymorphism (RFLP) analysis.


Genotypes of 345 individuals were determined simultaneously in a single PCR assay. Allele frequencies for our population were 6.8% for the TLR4 Asp299Gly polymorphism and 6.4% for the Thr399Ile polymorphism. Validation by RFLP analysis revealed a correct detection of all genotypes.


We have developed a novel method for the detection of the TLR4 Asp299Gly and Thr399Ile mutations, permitting rapid genotyping which should be useful for large-scale population studies as well as applicable for routine clinical testing.

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