MicroRNA-3148 modulates differential gene expression of the SLE-associated TLR7 variant

Abstract

Background: 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 x 10-10) [1]. 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. Method(s): 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. Result(s): 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 x 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). Conclusion(s): 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 x 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.