Determination of the TLR4 genotype using allele-specific PCR.

Despite significant advances in understanding the molecular basis of sepsis and its associated immunological response, sepsis remains a problem worldwide and is associated with a high mortality. Annually, septic shock, the most severe form of sepsis, causes the death of more than 100 000 people in the USA (3,4). Gram-negative bacteria are the most common pathogens associated with bacterial infections in diseases such as meningitis and represent more than 50% of septic shock-related bacteria. Endotoxin or lipopolysaccharide (LPS), the main component of the cell wall of Gram-negative bacteria, has been shown to elicit an inflammatory response that mimics all of the features described in septic shock (2). Recently, we have reported the association of two common polymorphisms in the human TLR4 gene with hyporesponsiveness to inhaled LPS (1). These mutations consist of single nucleotide polymorphisms (SNPs) in the coding region of the gene, both of which result in missense mutations. Analysis of additional patients revealed an association between TLR4 genotype and susceptibility to Gram-negative infections (unpublished data). The frequency of these mutations was between 6% and 10% in all tested Caucasian populations, making this mutation a common enough polymorphism to be of diagnostic value (1). Because of the potential usefulness of the TLR4 mutation in risk-stratifying patients vulnerable to endotoxin-induced disease, we developed a quick and reliable PCR-based genotyping assay. By altering the forward PCR primers, we were able to generate mutant TLR4-specific restriction sites that allow TLR4 genotyping using a simple restriction digest following the PCR amplification of genomic patient DNA. Study populations were made available through collaborations with several clinical facilities as indicated. For the graft-versus-host disease (GVHD) sample population, DNA samples were obtained from a previously described cohort of patients (n = 237) who received a hematopoietic stem cell transplantation (HSCT) from a human leukocyte antigen (HLA)-identical sibling (5). All patients received methotrexate and cyclosporine for GVHD prophylaxis and had either grade 0 or grades II-IV acute GVHD. Patients with grade I GVHD, patients with renal failure requiring dialysis, and patients without GVHD who died before day 80 after transplantation were excluded. DNA purification was done using a commercially available kit. For childhood asthma cases, we identified 120 cases with symptomatic asthma at study entry and 240 controls without persistent asthma at study entry. Subjects must have buccal cell DNA available to be eligible. Some of the genomic DNA used was derived from whole blood and purified using commercially available kits, such as Gentra Systems (Minneapolis, MN, USA). DNA purified using kits works very well in this assay. DNA purified as crude lysate using the NaOH lysis method will work as well, although, in general, a larger amount of sample is required to yield sufficient amounts of PCR product. PCR primers were designed to allow a distinction of wild-type and mutant TLR4 alleles based on the presence of restriction enzyme recognition sites. In both cases, the forward primer sequences were altered to generate a restriction site in the mutant allele. The following primers were used: for TLR4 Asp299Gly, forward 5′-GATTAGCATACTTAGACTACTACCTCCATG-3′ and reverse 5′-GATCAACTTCTGAAAAAGCATTCCCAC-3′, and for TLR4 Thr399Ile, forward 5′-GGTTGCTGTTCTCAAAGTGATTTTGGGAGAA-3′ and reverse 5′-ACCTGAAGACTGGAGAGTGAGTTAAATGCT3′. The underlined bases in both forward primers indicate the nucleotide altered to create a NcoI (TLR4 Asp299Gly) and HinfI (TLR4 Thr399Ile) restriction site, respectively. Reactions are set up using the AmpliTaq® PCR kit (Applied Biosystems, Foster City, CA, USA). Other PCR kits, such as the Advantage cDNA-PCR kit (Clontech Laboratories, Palo Alto, CA, USA) will work equally well. In a total reaction volume of 25 μL, 2.5 μL 10× PCR buffer, 20 pmol each primer (see above), 0.02 μg (0.2 μg for crude lysate) genomic DNA, 5 U Taq DNA polymerase, and 1 μL dNTP mixture (Clontech Laboratories) are combined. Reactions are run on a MJ Tetrad PTC225 Thermo Cycler (MJ Research, Waltham, MA, USA) using the following conditions: 95°C for 4 min, then 30 cycles of 95°C for 30 s, 55°C for 30 s, and 72°C for 30 s). Four microliters of the resulting PCR products are used for an overnight digest with the appropriate restriction enzyme, and digests are run out on a 3% NuSieve® GTG agarose gel (BMA, Rockland, ME, USA) to determine the TLR4 alleles. For the purpose of validating this method, 10 patient samples with different genotypes were sequenced directly using an ABI 377 sequencer (Applied Biosystems). Genomic DNA was amplified using forward 5′-TCTAGAGGGCCTGTGCAATT-3′ and reverse Benchmarks