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Microrna-Mediated Regulation Explains Allelic Risk of TLR7 Variant Predisposing to Systemic Lupus Erythematosis Disclosure All authors have no competing financial relationships to disclose. Yun Deng, Jian Zhao, Jennifer M. Grossman and Betty P. Tsao Division of Rheumatology, UCLA David Geffen School of Medicine Identification of TLR7 3’UTR SNP Associated with SLE Susceptibility in 9,274 Eastern Asians Introduction SLE: Excessive innate immune activation involving TLR7/8/9 and IFN-α an important pathogenic mechanism in the disease Increased Tlr7 Gene Dosage (Shen N, Fu Q, Deng Y, et al, PNAS, 2010;107:15838-15843) Autoimmunity In Lupus Mice Pisitkun P, et al. Science. 2006; Deane JA, et al. Immunity. 2007; Barrat FJ, et al. Eur J Immunol . 2007. No Strong Evidence For Common Copy Number Variations At Human TLR7-TLR8 Locus: 1. Real time PCR; 2. Genomic Southern and pulse field gel electrophoresis; 3. Reports from customized CGH arrays; 4. Database of Genomic Variants. Explore Functional TLR7 Single Nucleotide Polymorphisms Confirmation of the Association Between Rs3853839 and SLE Susceptibility in 13,339 non-Asian Ancestries Meta-analysis Showing Robust Association of rs3853839 with SLE in 22,613 individuals of Four Ancestries (Deng Y, Zhao J, et al, PLoS Genetics, 2013;9:e1003336) (Deng Y, Zhao J, et al, PLoS Genetics, 2013;9(2):e1003336) rs3853839 Trans-ethnic meta analysis Frequency 3,936 SLE vs. 3,491 Ctrls All 1,679 SLE vs. 1,934 Ctrls P meta OR (95%CI) Combined EA+AA+HS 7.5E-11 1.24 (1.18-1.34) Combined EA+AA+HS+AS 2.0E-19 1.25 (1.20-1.32) Sex EthnicityCase/Control Test Allele Case Control P OR (95%CI) EA 3,936/3,491 G 20.3% 17.2% 6.5E-06 1.23 (1.13-1.35) AA 1,679/1,934 G 19.8% 16.7% 1.1E-03 1.24 (1.09-1.41) HS 1,492/807 G 44.8% 37.3% 7.5E-04 1.26 (1.10-1.43) AS* 4,334/4,940 G 81.0% 77.0% 6.5E-10 1.27 (1.17-1.36) 1,492 SLE vs. 807 Ctrls 1 Risk Allele of rs3853839 TLR7 expression Risk Allele of rs3853839 mRNA Level in PBMCs: Slow degradation TLR7 expression Protein Level in PBMCs: Reporter assay: Xhol Renilla SV40 promoter Notl TLR7 3’UTR Firefly rs3853839 G/C RNA-containing autoantigens IRF7/IRF5 TLR7 IFN-α Explore whether differential miRNA binding could explain allelic risk of TLR7 for SLE. IFN-α inducible genes: MX1, Ly6E, IFIT1, IFIT3 Risk Allele of rs3853839 Risk Allele of rs3853839 TLR7 expression IFN response Reduced modulation by miRNA(s) TLR7 expression Only miR-3148 levels show an inverse correlation with TLR7 mRNA levels in PBMCs Co-transfection of each miRNA with TLR7 3’UTR reporter vector into HEK 293 cells SLE Control 2 Overexpression of miR-3148 in PBMCs leads to reduction in TLR7 mRNA expression PBMCs from healthy donors (n=15) miR-3148 mimic Non-target mimic 24h miR-3148 inhibitor Non-target inhibitor PBMCs stimulated with TLR7 agonist 24h Overexpression of miR-3148 in PBMCs results in a trend of decreased IFN-α production IFN-α production IFN-α inducible genes mRNA level TLR7 mRNA level mimic mimic NC inhibitor NC No miRNA transfection NC inhibitor NC No miRNA transfection miR-3148 miR-3148 Conclusion The TLR7 3’UTR SNP (rs3853839) was strongly associated with SLE susceptibility in populations of multiple ancestries. Among the 6 miRNAs predicted to target the sequences at/around rs3853839 at TLR7 3’UTR, miR-3148 and miR-2278 showed reduced modulation on the risk allele of rs3853839 which may confer decreased degradation of TLR7 transcripts, resulting in elevated TLR7 expression. miR-3148 levels in PBMCs showed an inverse correlation with TLR7 mRNA Acknowledgement UCLA Betty P. Tsao Yun Deng Jian Zhao Daisuke Sakurai Erika Magdangal Jennifer M. Grossman Rita M. Cantor Bevra H. Hahn levels. Overexpression of miR-3148 reduced the endogenous expression of TLR7 in PBMCs, resulting in a trend of decreased downstream production of type I IFNs; whereas inhibition of miR-3148 has an opposite effect. Thank You ! 3