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


METHODS
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.


RESULTS
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.


CONCLUSIONS
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.
van Rijn
Bas B
BB
Research Laboratory of the Department of Clinical Chemistry, University Medical Center Utrecht, The Netherlands. B.B.vanRijn@azu.nl
Roest
Mark
M
Franx
Arie
A
Bruinse
Hein W
HW
Voorbij
Hieronymus A M
HA
D000328:Adult; D019943:Amino Acid Substitution / Q000235:genetics; D004252:DNA Mutational Analysis / Q000379:methods*; D005260:Female; D005787:Gene Frequency; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D011110:Polymorphism, Genetic; D012150:Polymorphism, Restriction Fragment Length; D012680:Sensitivity and Specificity; D051197:Toll-Like Receptor 4 / Q000235:genetics*
D051197:Toll-Like Receptor 4