Computational protocol: Case-control study for colorectal cancer genetic susceptibility in EPICOLON: previously identified variants and mucins

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Protocol publication

[…] Selected SNPs were included in the previously identified category if they corresponded to genetic variants linked to CRC risk by previously published independent studies, or in the mucin gene family if located on genes encoding for mucins and GALNT proteins. Mucins are protein constituents of the mucous barrier highly glycosylated by GALNT proteins. One relevant previously identified SNP was studied in each of the following genes: ADH1C, APC, CCDN1, IL6, IL8, IRS1, MTHFR, PPARG, VDR and ARL11. For the mucin gene family, 1-2 SNPs were selected from each of the following genes: MUC7, MUC12, MUC13, MUC15, MUC16, MUC17, MUC19, MUC21, GALNT1, GALNTL2, GALNT10, GALNT14, GALNT4 and OVGP1. Mucin gene selection did not intentionally include GALTN12. This gene is located on the 9q22 region and it was studied in our candidate-gene approach for regions with positive linkage in CRC families (Abulí et al., manuscript in preparation). SNP selection in the mucin gene family was performed using Pupasuite, a web tool for the selection of genetic variants with potential phenotypic effect ( []. SNPs were always prioritized if they were coding, evolutionary conserved in mouse and SNP minor allele frequency (MAF) was above 5%. Other selected SNPs with a putative regulatory effect were in promoter, intronic or 3'-UTR regions. A complete list of SNP and genes analyzed in the present study is detailed in Table .High-throughput genotyping in EPICOLON cohorts was performed according to manufacturer's instructions with the TaqMan allelic discrimination and SNPlex™ systems (Applied Biosystems, Foster City, USA), and single-base primer extension chemistry matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) genotyping platform (Sequenom Inc., San Diego, USA). Genotyping in the MCC-Spain cohort was performed using the VeraCode technology (Illumina, San Diego, USA). Genotyping was performed at the Santiago de Compostela and Barcelona nodes of the Spanish National Genotyping Centre, and at the Genome Analysis Platform of the CIC-BioGUNE [...] As quality control, genotyping success was set above 90% for SNPs. Allelic frequency description and Hardy-Weinberg equilibrium test were performed using SNPator, a web-based tool offered by the Bioinformatics division of the Spanish National Genotyping Centre ( []. All SNPs analyzed had a genotype success rate > 90%. The genotype frequencies of all variants in the control population fitted the Hardy-Weinberg equilibrium (P > 0.01), supporting absence of genotyping artifacts. There was no sign of underlying population stratification in EPICOLON as tested by an independent study []. Genotype analysis was carried out using the SNPassoc R library []. Inter-group comparisons of genotype frequency differences were performed by regression analysis for codominant, dominant, recessive and log-additive models of inheritance. We estimated the crude odds ratio (OR) and their 95% confidence intervals (95% CIs). As expected, results did not change after sex and age adjustment. The best genetic or inheritance model was selected using the Akaike information criteria. To address the issue of multiple testing, we used Bonferroni correction (P = 0.0125 for four SNPs). Study power was estimated with CaTS software []. […]

Pipeline specifications

Software tools PupaSuite, SNPassoc
Application GWAS
Diseases Blood Platelet Disorders, Neoplasms, Colorectal Neoplasms