High-throughput identification of noncoding functional SNPs via type IIS enzyme restriction

Area of Science:

• Genomics
• Molecular Biology
• Genetics

Background:

• Genome-wide association studies (GWAS) identify numerous disease-associated noncoding variants.
• Distinguishing functional single-nucleotide polymorphisms (fSNPs) from non-functional ones within these loci remains challenging.
• Understanding the functional impact of these variants is crucial for elucidating disease mechanisms.

Purpose of the Study:

• To develop a high-throughput method for identifying functional single-nucleotide polymorphisms (fSNPs).
• To investigate the regulatory roles of disease-associated variants in specific genes and pathways.
• To bridge the gap between GWAS findings and biological mechanisms of disease.

Main Methods:

• Developed SNP-seq (single-nucleotide polymorphism sequencing), a high-throughput screen using type IIS enzymatic restriction to identify fSNPs.
• Coupled SNP-seq with flanking restriction enhanced pulldown (FREP) to identify protein-DNA interactions modulated by fSNPs.
• Applied the tandem SNP-seq/FREP approach to investigate disease-associated loci.

Main Results:

• Identified regulation of CD40 by three disease-associated fSNPs through four regulatory proteins (RBPJ, RSRC2, FUBP-1/TRAP150).
• Screened 27 loci associated with juvenile idiopathic arthritis, identifying 148 candidate fSNPs.
• Discovered two fSNPs regulating STAT4 via regulatory proteins SATB2 and H1.2.

Conclusions:

• The SNP-seq/FREP approach is effective for unbiased identification of functional variants.
• This method successfully links genetic risk loci to specific regulatory proteins and gene targets.
• Findings establish a powerful tool for dissecting the functional consequences of noncoding variants in disease.