Aging Institute, University of Pittsburgh, Pittsburgh, PA, 15219, USA.
School of Life Sciences, East China Normal University, Shanghai, China.
Nat Commun. 2020 Jul 3;11(1):3340. doi: 10.1038/s41467-020-17159-8.
GWAS cannot identify functional SNPs (fSNP) from disease-associated SNPs in linkage disequilibrium (LD). Here, we report developing three sequential methodologies including Reel-seq (Regulatory element-sequencing) to identify fSNPs in a high-throughput fashion, SDCP-MS (SNP-specific DNA competition pulldown-mass spectrometry) to identify fSNP-bound proteins and AIDP-Wb (allele-imbalanced DNA pulldown-Western blot) to detect allele-specific protein:fSNP binding. We first apply Reel-seq to screen a library containing 4316 breast cancer-associated SNPs and identify 521 candidate fSNPs. As proof of principle, we verify candidate fSNPs on three well-characterized loci: FGFR2, MAP3K1 and BABAM1. Next, using SDCP-MS and AIDP-Wb, we rapidly identify multiple regulatory factors that specifically bind in an allele-imbalanced manner to the fSNPs on the FGFR2 locus. We finally demonstrate that the factors identified by SDCP-MS can regulate risk gene expression. These data suggest that the sequential application of Reel-seq, SDCP-MS, and AIDP-Wb can greatly help to translate large sets of GWAS data into biologically relevant information.
GWAS 无法从连锁不平衡 (LD) 中的疾病相关 SNP 中识别功能 SNP(fSNP)。在这里，我们报告了三种连续方法的开发，包括 Reel-seq（调控元件测序）以高通量方式识别 fSNP、SDCP-MS（SNP 特异性 DNA 竞争下拉质谱）识别 fSNP 结合蛋白和 AIDP-Wb（等位基因不平衡 DNA 下拉-Western blot）检测等位基因特异性蛋白：fSNP 结合。我们首先应用 Reel-seq 筛选包含 4316 个乳腺癌相关 SNP 的文库，并鉴定出 521 个候选 fSNP。作为原理验证，我们在三个特征明确的基因座上验证候选 fSNP：FGFR2、MAP3K1 和 BABAM1。接下来，使用 SDCP-MS 和 AIDP-Wb，我们快速鉴定出多种调节因子，这些因子以等位基因不平衡的方式特异性结合 FGFR2 基因座上的 fSNP。最后，我们证明了 SDCP-MS 鉴定的因子可以调节风险基因的表达。这些数据表明，Reel-seq、SDCP-MS 和 AIDP-Wb 的顺序应用可以极大地帮助将大量 GWAS 数据转化为具有生物学意义的信息。
