Background/Purpose:

LBH (Limb-bud and heart development gene) is dysregulated in RA, with a SNP associated with increased RA risk located in an upstream enhancer. This mutation decreases LBH gene transcription in FLS and Lbh deficiency in the passive K/BxN arthritis model increases disease severity. To understand the functional significance of LBH SNPs, we investigated the individual and combined effects of additional SNPs on LBH regulation.

Methods:

FLS cultures were established from RA synovium obtained at the time of arthroplasty. Search of UCSC Genome Browser, NCBI dbSNP database and NIH GWAS Catalog identified two new LBH-associated SNPs, SNP1 and SNP2, that are modestly protective in RA. The two SNP1 (rs7579944) and SNP2 (rs1355208) are 300 bp apart in an intergenic region 9371 and 9071 bp upstream of the LBH TSS, respectively. Genomic DNA containing individual or combined RA Risk (Ref1 and -2) or RA Protective SNP1 and -2 alleles of LBH were cloned into minimal promoter pGL4.23-luciferase constructs. For luciferase assays, the control plasmids and single or combined Refs and SNPs plasmids (Ref1, SNP1, Ref2, SNP2, Ref1/Ref2 and SNP1/SNP2) were co-transfected into cultured RA FLS by nucleofection. Firefly luciferase activity was normalized to Renilla. Samples with minimal luciferase expression due to inadequate transfection efficiency were eliminated from the analysis.

Results:

Transfection of FLS with 5 of the 6 constructs increased transcriptional activity of the minimal promoter by 2.2-2.6 fold, with no significant differences between Ref1, SNP1, Ref2, SNP2, or SNP1/SNP2 from each other. Surprisingly, the increase in luciferase activity was significantly less with risk-associated Ref1/Ref2 combination compared with the protective SNP1/SNP2 construct (1.8±0.2 fold compared with 2.3±0.2 fold of the minimal promoter, p=0.0096, n=7). This pattern remained the same after stimulation with TGFß1 (1 ng/ml, 18 hr) or IL-1ß (2 ng/ml, 18 hr), even though these cytokines decrease the LBH gene expression in RA FLS. The NIH LDlink analysis tool showed that SNP1 and SNP2 have high linkage disequilibrium (D>0.99), with 44% of the population having the combined Refs and 39.7% having the combined RA protective SNPs.

Conclusion:

The combination of two RA Risk LBH-associated SNPs significantly decreased transcriptional activity compared to the individual SNPs or combination of the two corresponding protective alleles. These results correlate with the protective effect of LBH in arthritis and suggest that the mechanism of RA protection with the combination SNP1/SNP2 is higher LBH transcription compared with the risk alleles. Therefore the genetic risk conferred by individual LBH alleles is additive and underscores the importance of their combinatorial contributions to RA susceptibility.

« Back to 2018 ACR/ARHP Annual Meeting

ACR Meeting Abstracts - https://acrabstracts.org/abstract/additive-effects-of-functional-rheumatoid-arthritis-ra-lbh-risk-alleles-on-lbh-gene-transcription/