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Abstract Number: 18

Bridging the gap between GWAS and mechanism in juvenile idiopathic arthritis using a novel high-throughput experimental screen

Gang Li1, Marta Martinez-Bonet1, Di Wu2, Jing Cui3 and Peter A. Nigrovic1,4, 1Brigham and Women's Hospital, Boston, MA, 2University of North Carolina, Chapel Hill, Chapel Hill, NC, 3Rheumatology, Immunology, and Allergy, Brigham and Women's Hospital and Harvard Medical School, Boston, MA, 4Division of Immunology, Boston Children's Hospital, Boston, MA

Meeting: 2017 Pediatric Rheumatology Symposium

Keywords: genetics and juvenile idiopathic arthritis (JIA), GWAS

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Session Information

Date: Thursday, May 18, 2017

Session Title: Genetics and Pathogenesis Poster Breakout I

Session Type: Abstract Submissions

Session Time: 4:45PM-5:15PM

Background/Purpose:  Genome wide association studies (GWAS) have identified 27 non-HLA loci that convey risk for oligoarticular and seronegative polyarticular JIA. Most associated haplotypes bear no coding polymorphisms, implicating regulatory variants as a major driver of genetic predisposition. Unfortunately, predicting regulatory variants has proven extremely difficult, leaving obscure the mechanisms by which genes drive JIA risk.

Methods:  We devised an unbiased high-throughput screen that employs enzymatic restriction to identify genetic variants that allelically modulate the binding of regulatory proteins to DNA. In this method, termed Single Nucleotide Polymorphism-next generation sequencing (SNP-seq), each potentially regulatory variant is engineered into an individual DNA construct. A pool of constructs is then incubated with nuclear extract containing regulatory proteins such as transcription factors. PCR and next generation sequencing are then employed to amplify and quantitate sequences that bind nuclear proteins and are therefore spared from enzymatic degradation. To identify these bound proteins, we applied Flanking Restriction Enhanced Pulldown (FREP, Li…Nigrovic PLOS Genetics 2016;e1006292), an efficient method to employ SNP-containing sequences as bait constructs for protein identification by mass spectrometry.

Results:  We piloted SNP-seq + FREP on the CD40 locus, associated with rheumatoid arthritis, identifying three SNPs that determined expression of this co-stimulator via a previously unrecognized protein complex. We then performed a high-throughput screen across 1223 alleles of 608 SNPs in LD R2>0.8 with 27 JIA loci, using peripheral blood mononuclear cells as protein source, resulting in identification of 148 candidate regulatory variants. Confirmatory testing at the STAT4 locus established two novel regulatory DNA-protein interactions that regulate this T cell-associated gene in a manner implicated in JIA risk.

Conclusion:  We have developed a novel experimental strategy to identify genetic variants that modulate disease risk by altering the binding of transcription factors and other regulatory proteins. Application of this strategy to JIA has allowed us to begin to bridge the gap between GWAS and mechanism in this important but poorly-understood disease.


Disclosure: G. Li, None; M. Martinez-Bonet, None; D. Wu, None; J. Cui, None; P. A. Nigrovic, None.

To cite this abstract in AMA style:

Li G, Martinez-Bonet M, Wu D, Cui J, Nigrovic PA. Bridging the gap between GWAS and mechanism in juvenile idiopathic arthritis using a novel high-throughput experimental screen [abstract]. Arthritis Rheumatol. 2017; 69 (suppl 4). https://acrabstracts.org/abstract/bridging-the-gap-between-gwas-and-mechanism-in-juvenile-idiopathic-arthritis-using-a-novel-high-throughput-experimental-screen/. Accessed January 27, 2023.
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