Computational protocol: Variation in the ATP-binding cassette transporter 2 gene is a separate risk factor for Systemic Lupus Erythematosus within the MHC

Similar protocols

Protocol publication

[…] Tag SNPs were chosen for the TAP genes using Tagger (HapMap Phase II (release 19; NCBI build 35) with the following settings: “aggressive” (multimarker mode), r2 > 0.8 for allele and haplotype, allele frequency > 0.05, and HapMap CEU population Phase II data. These polymorphisms were genotyped by primer extension of multiplex products with detection by matrix-assisted laser desorption ionization-time of flight (MALDI-ToF) mass spectroscopy using a Sequenom platform. Only SNPs that met the following three criteria were used for association analysis: 1) Hardy-Weinberg P value ≥ 0.01, 2) more than 85% successful genotypes, and 3) no Mendelian errors. Pedigrees that showed Mendelian errors were eliminated, providing the 390 families used for the association analysis. Genotyping was performed for seven SNPs in TAP1 and ten SNPs in TAP2; one SNP in each gene failed to meet the above criteria and was rejected. In addition, 191 of these pedigrees had high-resolution four-digit HLA-DRB1 genotype data on the SLE affected offspring and at least one parent. DRB1 genotyping was performed by PCR-sequence-specific oligonucleotide (SSO) “linear array” methodology as previously described., Briefly, multiplex amplification of exon 2 of the DRB1 locus was performed with a set of 10 upstream PCR primers, corresponding to 10 sequence motifs in the first hypervariable region of the exon, and one common downstream primer. All primers were biotinylated, and resulting biotinylated PCR products were denatured and hybridized to a series of oligonucleotides, corresponding to known DRB1 sequence motifs, immobilized on a backed nylon membrane. Binding was detected by treatment of the membrane with streptavidin conjugated horseradish peroxidase followed by the colorimetric substrate tetramethlybenzidine. Probe binding was assessed using StripScan software (Roche Molecular Systems, Pleasanton, CA), and genotype calls were made using the Sequence Compilation and Rearrangement Evaluation (SCORE) software. Allele calls were reported at four-digit resolution. [...] Standard Transmission Disequilibrium Test (TDT) and haplotype estimation of the TAP1 and TAP2 genes were performed using Haploview v4.0 under default settings. The Pedigree Disequilibrium Test (PDT) was performed with UNPHASED using the EM option. The Odds Ratio (OR) for each SNP was calculated for the over-transmitted allele as the ratio of transmissions to non-transmissions. […]

Pipeline specifications

Software tools Tagger, Haploview, UNPHASED
Application GWAS
Diseases Lupus Erythematosus, Systemic
Chemicals Adenosine Triphosphate, Nucleotides