Functional Annotation And Experimental Validation Of Obesity GWAS Genes And SNPs

Obesity is a complex disease defined by BMI(Body mass index).Genome wide association study(GWAS)has become a major approach to discover genes and SNPs that contribute to complex diseases in the genome era.Up to May 2016,44 obesity GWASs and 10 meta-analyses had reported 163 susceptibility genes and 224 SNPs(p<5×10-8).The future task of obesity genetic study is to elucidate functional mechanisms through which these GWAS associated loci modulate obesity risk.Objectives of this study are to functionally annotate obesity GWAS-associated SNPs and genes,and to experimentally validate the effect of SNPs on the binding of miRNA,and to identify the target miRNAs of the TSC22D3 gene.A total of 4321 SNPs were identified as proxy SNPs of obesity GWAS-associated SNPs through SNAP.Twenty-three conservative SNPs were identified through VISTA and UCSC database.Two SNPs(rs2260000 and rs2229616)were detected to have long-range interaction signals with other loci by GWAS3D.RegulationDB analysis showed that 23 lead SNPs and 299 proxy SNPs with a score of less than 3.Some SNPs were identified as eQTLs of obesity associated genes ADCY3,MTCH2,SPl1 and KCTD15.Many SNPs located in the binding site of seven obesity related transcription factors(CEBPB,TCF7L2,STAT3,SPI1,GATA2,CREB1 and MEF2C)were found.Seven lead SNPs(rs3810291,rs2650492,rs10733682,rs6465468,rs879620,rs2531995 and rs7132908)and 28 proxy SNPs were predicted to affect the binding of miRNA by miRNASNP.Four lead SNPs(rs 12885454,rs591120,rs6265 and rs9925964)and 20 proxy SNPs were detected in IncRNAs by lncRNASNP,and some of them were predicted to influence the interaction between miRNA and lncRNA.Three lead SNPs(rs 11676272,rs6265 and rs591120)and 8 proxy SNPs were predicted to influence the phosphorylation of proteins by PhosSNP1.0.The network analysis of protein-protein interaction by STRING identified important hub proteins such as TMEM18,FTO,MC4R,SEC16B,GNPDA2,MTCH2,POC5,SH2B1,FAIM2,NEGR1,BDNF,KCTD15,CARK and TFAP2B.Obesity GWAS-associated genes mainly participated in metabolic process,and their main molecular function is catalytic activity.Neurotrophin signaling pathway,cell adhesion molecules,insulin secretion and vascular smooth muscle contraction were identified as significant enrichment pathways through David and STRING.Four miRNAs(miR-221,miR-143,miR-96 and miR-29b)were predicted to target TSC22D3 gene by TargetScan.Dual-luciferase reporter gene system was applied to validate the function of three SNPs(rs3810291,rs879620 and rs6465468),which were predicted to affect the binding of miRNA,and four miRNAs which were predicted to target TSC22D3.The results showed that rs6465468 may affect the binding of miR-101-5p with ASB4,and miR-29b may suppress the TSC22D3 expression.