Session Title: Genetics and Genomics of Rheumatic Disease II
Session Type: Abstract Submissions (ACR)
Background/Purpose: Identifying novel therapeutic targets for immune-mediated disease has become increasingly challenging. We used an unbiased method to integrate multiple “omics” methodologies and evaluate the overlap between RA-associated genes identified in genome-wide association studies (GWAS), genes that are differentially expressed or genes that are differentially methylated in RA fibroblast-like synoviocytes (FLS). This analysis narrowed the search to a limited number of genes and allowed us to focus on Engulfment and Cell Motility Protein-1 (ELMO1), a gene not previously considered as a therapeutic target. We show how this novel candidate gene plays a key role in the pathogenic behavior of RA FLS.
Methods: Data were from three types of genome-wide RA assays were integrated: (i) for sequence variation we used the NCBI GWAS database and extracted all gene that were mapped to SNPs that had been implicated in RA susceptibility (www.genome.gov/gwasstudies/); (ii) for gene expression we used public microarray datasets of RA, OA, and normal (NL) FLS (Gene Expression Omnibus Database); (iii) for DNA methylation we used a set of differentially methylated genes that we previously identified in RA, OA, and NL FLS (Genome Medicine 5:40 2013). For functional studies, passage 4 to 6 RA FLS were cultured in medium or PDGF and migration was measured using a scratch assay. Cell invasion was determined by measuring cell invasion into Matrigel-coated transwell plates. Rac1 activation was determined by measuring the Rac1-GTP bound form of Rac1 by Western blot. Gene knockdown was performed using the siRNA and Amaxa technology.
Results: The integrative analysis of the three unbiased genome-wide approaches identified 6 genes as different in RA compared to controls (OA and normal) in all datasets. Of the overlapping genes, we were particularly interested in the cytoplasmic engulfment protein, ELMO1, as it was differentially methylated in the promoter region, differentially expressed, and associated with RA due to an intron-6 polymorphism. This protein can bind to Rac1 in activated cells and potentially alter cell movement. We first confirmed that ELMO1 is expressed in RA FLS and synovium as determined by qPCR. siRNA knockdown was then performed, which decreased ELMO1 expression by >90% compared to control siRNA. ELMO1 deficiency decreased PDFG-induced FLS migration by 40% (p<0.02). In an invasion assay, ELMO1 siRNA markedly decreased invasion of PDGF stimulated RA FLS into Matrigel matrix compared with control siRNA. The mechanism ELMO1 function was determined by determining the effect of ELMO1 deficiency on Rac1 activation. These experiments showed that ELMO1 siRNA decreased peak PDGF-induced Rac1 activation by 80%.
Conclusion: Integrative analysis of multiple unbiased genome-wide datasets is a novel method to identify potential therapeutic targets. One previously unanticipated target, ELMO1, emerged as a candidate gene in RA. ELMO1 plays a critical role as a regulator of FLS migration and matrix invasion by activating Rac1. These data show how one can use integrative studies to identify and validate novel therapeutic targets.
D. L. Boyle,
G. S. Firestein,
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ACR Meeting Abstracts - https://acrabstracts.org/abstract/integrative-analysis-of-multiple-omics-technologies-reveals-a-novel-therapeutic-target-for-rheumatoid-arthritis-ra/