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We identified the G allele of TLR7 3'-UTR SNP (rs3853839) associated with increased TLR7 transcripts, a more pronounced IFN signature and risk for SLE in 9,274 Eastern Asians (Pcombined = 6.5 × 10-10) . The current study sought replication of SLE-associated SNP(s) in non-Asian ancestries and explored molecular mechanisms underlying an identified gene variant that affects TLR7 expression.
We conducted genotyping, imputation and association for 98 to 116 SNPs (varying among different ancestries) covering 80 kb of TLR7-TLR8 in European Americans (EA), African Americans (AA) and Hispanics enriched for the Amerindian-European admixture (HS). Haplotype-based conditional testing was conducted to distinguish independent association signals. Mantel-Haenszel testing was used in transancestral meta-analysis. Association of genotypes with TLR7 expression was examined using RT-PCR, flow cytometry and reporter assays. Pyrosequencing was used to measure allelic variations in TLR7 transcript levels.
The rs3853839 was confirmed as the only variant within TLR7-TLR8 exhibiting consistent and independent association with SLE in our transancestral fine-mapping (Pmeta = 7.5 × 10-11, OR (95% CI) = 1.24 (1.18 to 1.34)) in 13,339 subjects of EA (3,936 cases vs. 3,491 controls), AA (1,679 vs. 1,934) and HS (1,492 vs. 807) ancestries. PBMCs from normal G-allele carriers exhibited elevated levels of TLR7 mRNA (P = 0.01 in men and P = 0.02 in women) and protein (P = 0.009 in men and P = 0.038 in women). PBMCs from heterozygotes exhibited higher G/C allele ratios of TLR7 transcripts 4 hours after incubation with actinomycin D (inhibitor of transcription initiation) (P = 0.04), indicating slower degradation of G allele-containing transcript. The nonrisk allele, but not the risk allele, was predicted to match microRNA-3148 (miR-3148) at the second base in the binding site. Transcript levels of miR-3148 and TLR7 were inversely correlated in PBMCs from 16 SLE patients and 21 controls (R2 = 0.255, P = 0.001), suggesting miR-3148 modulating TLR7 expression. Overexpression of miR-3148 via transfection into HEK 293 cells led to a more than twofold reduction in luciferase activity driven by the TLR7 3'-UTR segment containing the nonrisk allele than that containing the risk allele (P = 0.001).
We identified and confirmed a genome-wide significant association between rs3853839 and SLE susceptibility in 22,613 subjects of Eastern Asian, EA, AA and HS ancestries (Pmeta = 6.4 × 10-19, OR (95% CI) = 1.26 (1.20 to 1.32)). Reduced modulation by miR-3148 confers slower degradation of the risk allele containing TLR7 transcript, resulting in elevated levels of gene products and a more robust type I IFN signature.