Computational And Functional Analysis Of Osteoporosis GWAS Associated Gene And SNP

Osteoporosis is a complex disease characterized by reduced bone mass and microarchitecture deterioration of bone tissues,thereby leading to loss of strength and increased risk of fractures.Osteoporosis is defined by bone mineral density(BMD).The heritability of BMD is about 60%-80%.Genome-wide association study(GWAS)is a powerful tool to identify suscepbility genes of common diseases.Until May 2015,a total of 25 GWAS articles had been published,reporting 107 genes and 129 SNPs(P<5×10-8)that are associated with osteoporosis(BMD).Future challenge is to understand the function and mechanism of these SNPs and genes action.One of our objectives is to conduct a computational and functional analysis of osteoporosis GWAS associated SNPs and genes.SOST was identified as a hub gene by protein-protein interaction analysis.SNP rs 1230399(T/C)in the SOST gene was significantly associated with BMD in our previous study.Computational analyses showed that the rs 123 0399 could affect the binding of transcription factor FOXA1.Another purpose of this study is to explore the function of the rs 1230399 polymorphism.1.Computational analyses of osteoporosis GWAS associated SNPs and genes(1)Conservation analysis of the osteoporosis GWAS-associated SNPs.Nine conservative SNPs(rs7741021、rs4355801、rs7071206、rs227425、rs9287237、rs2952559、rs3801387、rs4233949 and rs9533095)were identified through VISTA and UCSC database.(2)RegulomeDB annotation of osteoporosis GWAS-associated SNPs.A total of 119 GWAS lead SNP and proxy SNP with RegulomeDB score less than 3 may have potential regulation function.Some osteoporosis GWAS SNPs are the eQTLs of the genes FGFRL1,TNFRSF11B,LIN7C,ALDH7A1 and C6orf97,and are located at the binding sites of bone related transcript factors MEF2A,MEF2C,STAT3,THAP1,FOXA1,NFATC1,ESR1 and NFIC.(3)LncRNA analysis of osteoporosis GWAS associated SNP.Lead SNP-rs6894139 is located in lnc-RASA1-7 and influenced its binding with miR-369-3p and miR-8084.Proxy SNPS rs3806641 and rs1467561 is located in lnc-NAA50-1 and lnc-CKB-1 respectively,and rs3806641 can affect miR-6704 interaction with lnc-NAA50-1.(4)Long-rang interaction analysis of osteoporosis GWAS-associated SNPs.GWAS3D analysis showed that rs1463104,rs4729260 and rs2887571 have significant interaction signals with other loci over a long distance.(5)Interaction analysis of the SNP-miRNA-mRNA.GWAS lead SNPs rs884205 and rs1026364 and 14 proxy SNPS were predicted to affect miRNA and mRNA interaction.(6)Influence of GWAS associated SNPs on protein phosphorylation.GWAS Lead SNPs rs3755955,rs2707466,rs3736228 and proxy SNPs rs9866806,rs6831280,rs10416218 were predicted to affect the protein phosphorylation.(7)GO analysis of osteoporosis GWAS-associated genes.PANTHER analysis showed that osteoporosis GWAS-associated genes mainly participated in cell components(cell part,extracellular region,organelles,macromolecular complex and membrane),and mainly involved in biological processes(metabolic processes,cellular processes,biological regulation and development processes)and molecular functions(binding,catalytic activity,receptor activity and binding activity of nucleic acid and transcription factor).DAVID analysis identified 23 biological processes,4 molecular functions and 4 cell components.(8)Signaling pathway enrichment analysis.Significant enrichment signaling pathways of osteoporosis susceptibility genes are Wnt signaling pathway,Basal cell carcinoma and Hedgehog signaling pathway.(9)Protein-protein interactional network analysis.Hub genes(proteins)RUNX2,SP7,TNFRSF11B,LRP5,ESR1,SOST and DKK1 were identified by STRING2.Functional study of SOST rs1230399(1)Functional analysis of SOST rs1230399:When 293T,Saos-2 and U2-OS cells were transfected by the rs1230399-T and rs1230399-C carrier,dual luciferase reporter gene assays showed that the luciferase activity of recombinant vector T decreased significantly compared to recombinant vector C.When transfected cell by FOXA1 interference carrier and overexpression vector,the luciferase report gene activity of rs1230399-T+FOXA1.shRNA was higher than the luciferase activity of rs1230399-C+FOXA1.shRNA group and rs1230399-T group.The luciferase report gene activity of rs1230399-T +FOXA1 overexpression group in all of the three cells was lower than the luciferase activity of rs1230399-C+FOXA1 overexpression group.The biotin-labeled pull down experiment showed that the probe with the T allele bind FOXA1 antibody more strongly than the probe with the C allele.(2)Effects of FOXA1 on SOST expression.The expression of SOST mRNA and protein elevated significantly when FOXA1 was knocked down and reduced when FOXA1 overexpressed.(3)Influence of hormone on the FOXA1 expression.The FOXA1 expression slightly increased when 293 T cells were stimulated with 10 nM estrogen for 15h,and significantly elevated when stimulated with 50 nM PTH for 16h.Our results revealed a novel mechanism that rs1230399 may regulate SOST expression and was associated with BMD by influencing FOXA1 binding.