Background/Purpose: Fetuses exposed to maternal anti-Ro60 antibodies can develop cardiac conduction abnormalities and life threatening cardiomyopathy; manifestations of neonatal lupus (NL). Recent data support an injury model in which immune complexes (IC) comprised of Ro60, ssRNA, and anti-Ro60 engage Toll-like receptors (TLR) and promote secretion of proinflammatory and profibrotic factors.  To identify risk genes related to TLR-dependent fibrosis, a novel strategy was designed based on: 1) gene expression of macrophages stimulated by Ro60-related hY3 ssRNA as a proxy of the in utero environmental contribution and 2) the association of genetic variation with cardiac NL. This is the first approach to employ environmental and genetic determinants to identify the association of risk genes in anti-Ro60-mediated injury.

Methods:  Human macrophages obtained from PBMCs were transfected with hY3 or cultured with IC containing Ro60, anti-Ro60,and hY3 to ligate TLR7 with or without IRS661, a specific TLR7 inhibitor. Affymetrix HG-U133A 2.0 microarray analysis using RNA isolated from the test conditions (n=6) was employed.  Selection was prioritized to expression of genes common to both hY3 and IC stimulation in which the copy number increased by 50%. Ingenuity Pathway Analysis was used to generate a list of genes with fibrotic functions.  Variants from our previously published genome-wide association study (GWAS) were used to test for an enrichment of association in these genes. This list was then compared to the overexpressed transcripts, and shared genes were ranked using a weighted composite score with a focus on the TLR7 pathway (ratio of transcript copy number for IC/IC+IRS661, high to low, and by the P-value of the variant, most significant to least).

Results:   In hY3 and IC stimulated conditions 1184 shared genes were upregulated of which 1110 and 891 were reduced by 50% with IRS661 co-treatment, respectively. Among these, there was an enrichment of genes known to be related to inflammation and fibrosis.  Based on our previously published GWAS, 327 fibrotic genes showed a significant enrichment of associations with cardiac NL (P=2.27×10^-9).  Of these genes, 20 were overexpressed in the macrophage array in a TLR7-dependent manner.  To select candidates with a potential functional role in mediating cardiac injury, the candidate list was narrowed to three genes based on a ranking of a composite score. The risk gene with the highest score was the relaxin receptor 1 (RXFP1) (transcript copy number (IC/(IC+IRS661)) ratio=21.2 and rs10517692 P=0.035), a G-protein coupled receptor that binds relaxin and plays a role in collagen and connective tissue remodeling. Key inflammatory mediators included C5 (ratio=6.4, rs2269066 P=0.008) and TNF (ratio=4.1, rs2844482 P=0.004). The latter was previously reported to have both genetic variation and biologic relevance associated with cardiac NL.

Conclusion:  These data support the applicability of a novel model in which causal alleles can be identified by combining expression and association datasets to delineate the molecular mechanisms by which anti-Ro60 antibody mediates cardiac injury. The identification of RXFP1 and the further confirmation of TNF provide important targets for consideration.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/modeling-environmental-and-genetic-determinants-to-identify-the-association-of-risk-genes-in-anti-ro60-mediated-injury-relaxin-receptor-i-and-tumor-necrosis-factor/