A sequential methodology for the rapid identification and characterization of breast cancer-associated functional SNPs

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adaptor Proteins, Signal Transducing / genetics
  • Blotting, Western
  • Breast Neoplasms / genetics*
  • Breast Neoplasms / pathology
  • Cell Line, Tumor
  • Female
  • Gene Expression Regulation, Neoplastic
  • Genetic Predisposition to Disease / genetics*
  • Genome-Wide Association Study / methods*
  • Humans
  • Linkage Disequilibrium
  • MAP Kinase Kinase Kinase 1 / genetics
  • MCF-7 Cells
  • Mass Spectrometry / methods
  • Polymorphism, Single Nucleotide*
  • Receptor, Fibroblast Growth Factor, Type 2 / genetics
  • Regulatory Sequences, Nucleic Acid / genetics
  • Sequence Analysis, DNA / methods*


  • Adaptor Proteins, Signal Transducing
  • BABAM1 protein, human
  • Receptor, Fibroblast Growth Factor, Type 2
  • MAP Kinase Kinase Kinase 1