High Throughput Identification of Non-Coding Functional SNPs via Type IIS Enzyme Restriction

Genome wide association studies have identified many disease-associated non-coding single nucleotide polymorphisms, but cannot distinguish functional SNPs (fSNPs) from others that reside incidentally within risk loci. To address this challenge, we developed an unbiased high-throughput screen that employs type IIS enzymatic restriction to identify fSNPs that allelically modulate the binding of regulatory proteins. We coupled this approach, termed SNP-seq, with flanking restriction enhanced pulldown (FREP) to identify regulation of CD40 by 3 disease-associated fSNPs via 4 regulatory proteins, RBPJ, RSRC2, and FUBP-1/TRAP150. Applying this approach across 27 loci associated with juvenile idiopathic arthritis, we identified 148 candidate fSNPs, including two that regulate STAT4 via the regulatory proteins SATB2 and H1.2. Together, these findings establish the utility of tandem SNP-seq/FREP to bridge the gap between GWAS and disease mechanism.