Key Laboratory of Biomedical Information Engineering of Ministry of Education, and Institute of Molecular Genetics, School of Life Science and Technology, Xi'an Jiaotong University, Shaanxi, Xi'an, People's Republic of China.
J Bone Miner Res. 2018 Jul;33(7):1335-1346. doi: 10.1002/jbmr.3419. Epub 2018 May 17.
RANKL is a key regulator involved in bone metabolism, and a drug target for osteoporosis. The clinical diagnosis and assessment of osteoporosis are mainly based on bone mineral density (BMD). Previous powerful genomewide association studies (GWASs) have identified multiple intergenic single-nucleotide polymorphisms (SNPs) located over 100 kb upstream of RANKL and 65 kb downstream of AKAP11 at 13q14.11 for osteoporosis. Whether these SNPs exert their roles on osteoporosis through RANKL is unknown. In this study, we conducted integrative analyses combining expression quantitative trait locus (eQTL), genomic chromatin interaction (high-throughput chromosome conformation capture [Hi-C]), epigenetic annotation, and a series of functional assays. The eQTL analysis identified six potential functional SNPs (rs9533090, rs9594738, r8001611, rs9533094, rs9533095, and rs9594759) exclusively correlated with RANKL gene expression (p < 0.001) at 13q14.11. Co-localization analyses suggested that eQTL signal for RANKL and BMD-GWAS signal shared the same causal variants. Hi-C analysis and functional annotation further validated that the first five osteoporosis SNPs are located in a super-enhancer region to regulate the expression of RANKL via long-range chromosomal interaction. Particularly, dual-luciferase assay showed that the region harboring rs9533090 in the super-enhancer has the strongest enhancer activity, and rs9533090 is an allele-specific regulatory SNP. Furthermore, deletion of the region harboring rs9533090 using CRISPR/Cas9 genome editing significantly reduced RANKL expression in both mRNA level and protein level. Finally, we found that the rs9533090-C robustly recruits transcription factor NFIC, which efficiently elevates the enhancer activity and increases the RANKL expression. In summary, we provided a feasible method to identify regulatory noncoding SNPs to distally regulate their target gene underlying the pathogenesis of osteoporosis by using bioinformatics data analyses and experimental validation. Our findings would be a potential and promising therapeutic target for precision medicine in osteoporosis. © 2018 American Society for Bone and Mineral Research.
RANKL 是一种参与骨代谢的关键调节因子，也是骨质疏松症的药物靶点。骨质疏松症的临床诊断和评估主要基于骨密度 (BMD)。先前的全基因组关联研究 (GWAS) 已经确定了多个位于 RANKL 上游 100kb 以上和 AKAP11 下游 65kb 处的 13q14.11 上的基因间单核苷酸多态性 (SNP)，这些 SNP 与骨质疏松症有关。这些 SNP 是否通过 RANKL 发挥作用尚不清楚。在这项研究中，我们进行了综合分析，结合表达数量性状基因座 (eQTL)、基因组染色质相互作用 (高通量染色体构象捕获 [Hi-C])、表观遗传注释和一系列功能测定。eQTL 分析确定了六个潜在的功能性 SNP(rs9533090、rs9594738、r8001611、rs9533094、rs9533095 和 rs9594759)，它们与 13q14.11 上的 RANKL 基因表达仅呈显著相关 (p<0.001)。共定位分析表明，RANKL 和 BMD-GWAS 信号的 eQTL 信号共享相同的因果变异。Hi-C 分析和功能注释进一步验证了前五个骨质疏松症 SNP 位于一个超级增强子区域，通过长距离染色体相互作用调节 RANKL 的表达。特别是，双荧光素酶报告基因实验显示，超级增强子中 rs9533090 所在区域具有最强的增强子活性，rs9533090 是一个等位基因特异性调控 SNP。此外，使用 CRISPR/Cas9 基因组编辑删除 rs9533090 所在区域，显著降低了 RANKL 在 mRNA 水平和蛋白水平的表达。最后，我们发现 rs9533090-C 可以强有力地募集转录因子 NFIC，从而有效地提高增强子活性并增加 RANKL 的表达。总之，我们通过生物信息学数据分析和实验验证，提供了一种可行的方法来识别调节性非编码 SNP，以通过远程调节其潜在的骨质疏松症发病机制下的靶基因。我们的研究结果为骨质疏松症的精准医学提供了一个有潜力和有前途的治疗靶点。
