Integrate Genomic And Transcriptomic Data To Identify Novel Osteoporosis-related Genes

Objective: Osteoporosis(OP)is the most common skeletal disorder in the world,characterized by reducing of bone mass,increasing of bone fragility,and destruction of bone microstructure,which will increase the risk of fracture.Though genome wide association studies(GWASs)have identified hundreds of genetic variants associated with bone mineral density(BMD)and fracture,it remains a challenge to interpret their biological functions and underlying biological mechanisms.To identify novel osteoporosis-associated loci and genes,and further research related biological mechanisms and molecular pathways,we performed a Conditional false discovery rate(c FDR)study and a Transcriptome-wide association study(TWAS)study,respectively.Methods: In the c FDR study,we conducted a c FDR analysis to combine the GEFOS total Body BMD(TB＿BMD)GWAS(n=66,628)and the UKBB estimated BMD from quantitative heel ultrasounds(e BMD)GWAS(n=426,824)and detected some osteoporosis-associated single nucleotide polymorphisms(SNPs).Then we used webbased annotation tool FUMAGWAS to map these SNPs to their corresponding candidate effect genes according to genome position,expression quantitative trait loci(e QTLs)and3D chromatin interaction.Next,we performed differentially expressed genes(DEGs)analysis,using RNA-seq data from 122 Caucasian females(63 individuals with high BMD and 59 individuals with low BMD)to identify DEGs.Last,we performed the GO and KEGG pathways enrichment analysis to the candidate effect genes mapped by the SNPs detected in c FDR anaysis and 1476 significantly DEGs separately and identified the overlapping genes and pathways across them.In the TWAS study,we performed S-Predi Xcan and S-Multi Xcan to integrate diverse expression quantitative trait loci(e QTLs)and splicing QTLs(s QTLs)data with several powerful GWASs datasets to identify novel candidate genes associated with osteoporosis.Here,we conducted a TWAS for TB＿BMD(n = 66,628 for discovery and7,484 for validation)and fracture(53,184 fracture cases and 373,611 controls for discovery and 37,857 cases and 227,116 controls for validation),respectively.We also conducted multi-SNP-based summarized mendelian randomization(SMR)analysis to assess the causal effect of the genes detected by TWAS to the traits.We also performed gene function annaotation analyses to the genes identified in TWAS and SMR,including protein-protein interaction(PPI)network and GO/KEGG functional enrichment analyses.Results: In the c FDR analysis,we totally identified 994 pleiotropic SNPs associated with osteoporosis.These SNPs were then mapped to 2050 candidate effect genes according to genome position,e QTLs and 3D chromatin interaction.Among them,some SNPs did not reach the genome-wide significance level in the original GWASs,which were tend to be overlooked in previous study,such as rs7517415 and rs11207730.We found both of these two SNPs have effects to the local gene TRABD2 B,which playes an important role in osteoporosis through the Wnt/β-catenin pathway.In DEGs analysis,we totally detected 1476 significantly DEGs,including 42 up-regulated genes and 1434down-regulated genes respectively.Among them,36 genes were mapped by the pleiotropic SNPs detected in c FDR analysis,such as AASS、IYD and CYP3A43.We obtained 2 common enriched GO terms in these two groups of genes: GO＿BP: Response to lipid and GO＿MF: Cofactor binding.In the TWAS study,we totally detected 82 genes significantly associated with TB＿BMD or fracture through expression or RNA splicing,and revealed that 74 of these significant genes may have potential causal effects on TB＿BMD or fracture in at least one specific tissue,including 63 genes reported in previous GWAS or TWASs for osteoporosis,such as ING3,CPED1 and WNT16,as well as 11 novel genes,such as DBF4 B,GRN,TMUB2 and UNC93B1.Among the 74 genes,39 genes formed a significant PPI network,and 4novel genes(DBF4B,GRN,TMUB2 and UNC93B1)detected in our study were directly connected to the known genes,such as ESPL1,NAGLU and TCIRG1.We identified 15 biological processes(BP)in GO and 5 pathways in KEGG through functional enrichment analysis,including some BP or pathyways known to related with osteoporosis,such as Wnt signaling pathway,osteoblast differentiation and skeletal system development.Conclusions: We validated some known osteoporosis-associated genes,such as TRABD2 B,ING3,CPED1,and WNT16,and molecular pathways which play important roles in osteoporosis,such as the Wnt signaling pathway through c FDR and TWAS.At the same time,we also identified some new osteoporosis related genes,such as DBF4 B,GRN,TMUB2 and UNC93B1.We hope that our findings could enhance the interpretation of the existing GWASs data for osteoporosis,provide new clues for revealing the pathogenesis of osteoporosis and may contribute to the future development of gene-based intervention/prevention/prediction for osteoporosis.