Computational protocol: Polymorphisms in FFAR4 (GPR120) Gene Modulate Insulin Levels and Sensitivity after Fish Oil Supplementation

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Protocol publication

[…] The International HapMap Project SNP database, based on the NCBI B36 assembly Data Rel 28. phase II + III, build 126, was used to identified FFAR4 tagged SNPs []. We added 500-kilo base pairs downstream of FFAR4 and 2500-kilo base pairs upstream to cover the 5’UTR and 3’UTR regions. Gene Tagger procedure in Haploview V4.2 was used to determine FFAR4 tagged SNPs using a pairwise tagging (r2 ≥ 0.8) and a minor allele frequency ≥5%. Subsequently, we used the LD Plot procedure in Haploview V4.2 to examine the linkage disequilibrium out of the twelve FFAR4 SNPs (). Genomic DNA was prepared using the SIGMA GenElute Gel Extraction Kit (Sigma-Aldrich Co. St. Louis, MO, USA). FFAR4 tagged SNPs (rs17484310, rs11187515, rs1414929, rs12415204, rs12219199, rs2065875, rs7081686, rs11187527, rs11187529, rs11187534, rs11187537, rs17108973) have been genotyped in 210 individuals using validated primers and TaqMan probes (Life Technologies Corporation, Burlington, ON, Canada) []. DNA was mixed with TaqMan Universal PCR Master Mix (Life Technologies Corporation), with a gene-specific primer and with probe mixture in a final volume of 10 μL (predeveloped TaqMan SNP Genotyping Assays). Genotypes were determined and analyzed using a 7500 RT-PCR System and ABI Prism SDS version 2.0.5 (Life Technologies Corporation). [...] The first 30 subjects who finished the intervention had their transcript expression measured in peripheral blood mononuclear cells using the Human-6 v3 Expression BeadChips (Illumina, San Diego, CA, USA). Transcriptomic analyses have been described in a previous paper []. The Berkeley Drosophilia Genome Project splice site prediction tool was used for RNA splicing analyses []. The SNP Annotation and Proxy Search (SNAP) online tool was used to evaluate LD between tagged and other non-tagged SNPs in the Northern Europeans from Utah (CEU) population []. The r2 was 80% and the distance limit was 500 kb. GWAS catalog was then used to verify if tagged and non-tagged SNPs have been previously identified in a GWAS []. Finally, the Transcription factor Affinity Prediction (TRAP) web tool for two sequences was used to evaluate transcription factor binding affinity differences between the alleles of tagged SNPs [,,]. […]

Pipeline specifications

Software tools Tagger, Haploview, SNAP, TRAP
Databases International HapMap Project GWAS Catalog
Application GWAS
Chemicals Fatty Acids, Eicosapentaenoic Acid