BACKGROUND: Pathogen recognition receptors such as Toll-like receptors (TLRs), which recognize pathogen-associated molecular patterns, lead to the activation of innate immunity. Genetic variations in these receptors may lead to an altered host immune response to pathogens. METHODS: We developed homogeneous fluorescence-based PCR assays as well as matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) genotyping assays to detect TLR4 polymorphisms. These assays were compared with restriction fragment length polymorphism (RFLP) analysis. Peripheral blood monocytes from donors with differing genotypes were prepared and exposed to bacterial products in vitro. The abundance of mRNAs of the proinflammatory cytokines interleukin (IL)-1 beta, IL-6, and tumor necrosis factor-alpha from these monocytes were monitored by real-time reverse transcription-PCR. RESULTS: By our homogeneous PCR method, the allele frequencies were 5.6% for the TLR4 Asp299Gly and 6.0% for the TLR4 Thr399Ile polymorphism in 116 healthy German Caucasians. Nine incorrect genotype calls were detected in the RFLP analysis and two in the TaqMan genotype analysis. MALDI-TOF-MS allowed clear detection of all TLR4 alleles. Monocytes from donors homozygous for the TLR4 mutant alleles Asp299Gly and Thr399Ile were lipopolysaccharide hyporesponsive and exhibited median effective concentrations (EC(50)s) approximately fourfold higher than those of monocytes carrying wild-type or heterozygous alleles. In contrast, a TLR2 agonist elicited similar responses in monocytes irrespective of the TLR4 genotype. CONCLUSIONS: Homogeneous fluorescence-based PCR assays provide a specific and sensitive method for high-throughput genotyping of TLR4 mutations. The newly developed PCR and MALDI-TOF-MS assays may be useful to evaluate the presence of TLR4 polymorphisms in patients to predict susceptibility to bacterial infection.
BACKGROUND: Pathogen recognition receptors such as Toll-like receptors (TLRs), which recognize pathogen-associated molecular patterns, lead to the activation of innate immunity. Genetic variations in these receptors may lead to an altered host immune response to pathogens. METHODS: We developed homogeneous fluorescence-based PCR assays as well as matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) genotyping assays to detect TLR4 polymorphisms. These assays were compared with restriction fragment length polymorphism (RFLP) analysis. Peripheral blood monocytes from donors with differing genotypes were prepared and exposed to bacterial products in vitro. The abundance of mRNAs of the proinflammatory cytokines interleukin (IL)-1 beta, IL-6, and tumor necrosis factor-alpha from these monocytes were monitored by real-time reverse transcription-PCR. RESULTS: By our homogeneous PCR method, the allele frequencies were 5.6% for the TLR4Asp299Gly and 6.0% for the TLR4Thr399Ile polymorphism in 116 healthy German Caucasians. Nine incorrect genotype calls were detected in the RFLP analysis and two in the TaqMan genotype analysis. MALDI-TOF-MS allowed clear detection of all TLR4 alleles. Monocytes from donors homozygous for the TLR4 mutant alleles Asp299Gly and Thr399Ile were lipopolysaccharide hyporesponsive and exhibited median effective concentrations (EC(50)s) approximately fourfold higher than those of monocytes carrying wild-type or heterozygous alleles. In contrast, a TLR2 agonist elicited similar responses in monocytes irrespective of the TLR4 genotype. CONCLUSIONS: Homogeneous fluorescence-based PCR assays provide a specific and sensitive method for high-throughput genotyping of TLR4 mutations. The newly developed PCR and MALDI-TOF-MS assays may be useful to evaluate the presence of TLR4 polymorphisms in patients to predict susceptibility to bacterial infection.
Authors: Bart Ferwerda; Matthew Bb McCall; Karlijn Verheijen; Bart-Jan Kullberg; André Jam van der Ven; Jos Wm Van der Meer; Mihai G Netea Journal: Mol Med Date: 2008 May-Jun Impact factor: 6.354