Computational protocol: Genetic variation and expression levels of tight junction genes identifies association between MAGI3 and inflammatory bowel disease

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[…] Blocks of genetic linkage disequilibrium in the region of MAGI3 and PTPN22 were identified using data from HapMap (available at: www.hapmap.org; HapMap Data Rel. 28 Phase II + III, August10) (Fig. ). The blocks were visualized using Haploview and the algorithm by Gabriel et al. [] based on 95% confidence bounds on D’ (Haploview version 4.2, available at: www.broad.mit.edu/haploview). [...] DNA was isolated from buffy coat or whole blood (EDTA blood) using the MagNA Pure LC DNA Isolation Kit and MagNA Pure extraction robot (Roche, Basel, Switzerland). In patients where no DNA from blood was available, the genotyping was performed using DNA isolated from intestinal biopsies (isolation previously described []). Allelic discrimination was carried out using either the TaqMan OpenArray system or real-time PCR, as well as TaqMan SNP genotyping assays (Applied Biosystems, Foster City, CA) (Additional file : Table S1).Allelic discrimination using TaqMan OpenArray Genotyping Plates, TaqMan OpenArray Genotyping Master Mix, GeneAmp PCR System 9700, and OpenArray NT Imager (Applied Biosystems) was in accordance with the manufacturer’s recommendation. Genotype data were analyzed using OpenArray SNP Genotyping Analysis Software version 1.0.3 and TaqMan Genotyper Software v.1.3 (Applied Biosystems).The allelic discrimination using real-time PCR was performed in a total reaction volume of 10 μL, consisting of TaqMan SNP genotyping assays and TaqMan Genotyping Master Mix using either the 7500 Fast Real-Time PCR System (Applied Biosystems) or the CFX96 Real-time System, C1000 Touch Thermal Cycler (Bio-Rad Laboratories Inc., Hercules, CA). The genotype data was analyzed using 7500 Software version 2.0.6 (Applied Biosystems) or Bio-Rad CFX Manager 3.1 (Bio-Rad Laboratories Inc.). [...] Allelic odds ratios (OR) and p-values, based on chi-squared (χ2) tests, were calculated using JMP Genomics 6.0 (JMP Genomics 6.0; SAS Institute Inc., Cary, NC). Deviation from Hardy-Weinberg equilibrium was analyzed using the exact test implemented in Haploview version 4.2 (available at: http://www.broad.mit.edu/haploview). For these statistical tests, p < 0.05 was considered significant.Genes with significant association to IBD, CD, or UC were further analyzed. Group differences in gene expression were investigated using Kruskal-Wallis ANOVA or Mann–Whitney U test (Statistica 12; StatSoft Inc., Tulsa, OK). Gene expression levels were investigated in relation to genotype using Spearman’s rank correlation test (Statistica version 12.7; StatSoft Inc., Tulsa, OK) and also in relation inflammatory status, phenotype, and ongoing medical treatment using logistic regression (Statistica version 12.7; StatSoft Inc.). The analyses of the group differences of gene expressions as well as the logistic regressions were performed using ΔCT values. Box plots were created using Statistica version 12.7 (StatSoft Inc.). A Bonferroni adjusted p < 0.008 (based on the number of analyzed genes; n = 6) was considered significant for statistical analysis based on gene expression levels. […]

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