Computational protocol: Dectin-1 and DC-SIGN Polymorphisms Associated with Invasive Pulmonary Aspergillosis Infection

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Protocol publication

[…] Twenty-seven tagging/functional SNPs within DC-SIGN, Dectin-1, Dectin-2, CCL2 and CCR2 were selected to genotype the entire panel of individuals (). The aim of the SNP tagging was to identify a set of SNPs that efficiently tags all the known SNPs while the functional approach was used to determine the net impact of potentially functional variants within DC-SIGN, Dectin-1, Dectin-2, CCL2 and CCR2 genes on IPA risk. Tagging SNPs were selected using Haploview Tagger program (http://www.broad.mit.edu/mpg/haploview/; http://www.broad.mit.edu/mpg/tagger/) and a pairwise tagging with a minimum r2 of 0.8. In this selection we forced the inclusion of the DC-SIGNrs4804803, CCL2rs1024610 and CCL2rs1024611 polymorphisms as their functionality has been suggested –. Genomic DNA was extracted from peripheral blood mononuclear cells (PBMCs) Qiagen Mini Kit (Qiagen, Valencia, CA, USA). Genotyping of DC-SIGN, Dectin-1, Dectin-2, CCL2 and CCR2 polymorphisms was carried out using KASPar assays (KBiosciences, Hoddesdon, Hertfordshire, UK) in a 384-well plate format (Applied Biosystems, Foster City, CA, USA) where hematological patient samples were randomly distributed. KASPar reactions were performed using KASPar assay mix (containing probes) and KASPar kit containing 2× Reaction Mix and MgCl2 (50 mM). PCR conditions were: denaturation at 94°C for 15 min, 10 cycles of denaturation at 94°C for 10 sec, annealing at 57°C for 5 sec and elongation at 72°C for 10 sec and 20 cycles of denaturation at 94°C for 10 sec, annealing at 57°C for 20 sec and elongation at 72°C for 40 sec. Recycling conditions were 94°C for 10 sec, annealing and elongation at 60°C for 60 sec. PCR products were analyzed with the ABI Prism 7900HT detection system using the SDS 2.4 software (Applied Biosystems). For internal quality control, about 5% of samples were randomly selected and included as duplicate. Concordance between the original and the duplicate samples for the 27 SNPs analyzed was ≥99.5%. Call rates for all SNPs were ≥97.8% with the exception of the Dectin-1rs11053599 SNP with a call rate of 94.5%. [...] We used the Web-based tool FastSNP available at http://fastsnp.ibms.sinica.edu.tw for predicting the functional significance of the SNPs associated with IPA infection. FastSNP utilizes information from another online program PolyPhen (http://www.bork.embl-heidelberg.de/PolyPhen/) and from four different web resources (TFSearch, ESEfinder, Rescue-ESE and FAS-ESS) to determine whether SNPs are located at exonic splicing regulatory sites, cause a non-conservative amino acid change or whether they alter the transcription factor-binding site of a gene (for instance, acting as intronic enhancer). The score was given by this tool on the basis of levels of risk with a ranking of 0 (no effect), 1 (very low), 2 (low), 3 (medium), 4 (high), or 5 (very high). […]

Pipeline specifications

Software tools Haploview, FastSNP, PolyPhen, TFSearch, ESEfinder, RESCUE-ESE
Applications WGS analysis, GWAS
Organisms Homo sapiens
Diseases Pulmonary Aspergillosis, Invasive Pulmonary Aspergillosis