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- Wiley, MM, et al.
(författare)
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FUNCTIONAL EVALUATION OF THE SJOGREN'S SYNDROME AND SYSTEMIC LUPUS ERYTHEMATOSUS DDX6-CXCR5 RISK INTERVAL
- 2020
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Ingår i: ANNALS OF THE RHEUMATIC DISEASES. - : BMJ. - 0003-4967 .- 1468-2060. ; 79, s. 89-90
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- Sjögren’s Syndrome (SS) and Systemic Lupus Erythematosus (SLE) are distinct chronic, complex autoimmune diseases with shared characteristics such as autoantibodies, heightened interferons, and polyarthritis. SS and SLE genome-wide association studies (GWAS) report strong associations with theDDX6-CXCR5risk interval. DDX6 suppresses interferon stimulated gene expression and CXCR5 regulates T cell functions implicated in autoimmunity.Objectives:To identify functional variants that impact regulation in theDDX6-CXCR5interval.Methods:Fine-mapping was done using ImmunoChip data from 3785 SLE, 1916 SS cases and 6893 population controls of European ancestry that were imputed and tested for SNP-trait association. Bayesian statistics assigned posterior probabilities to SNPs and defined a credible set of risk variants. Bioinformatic analyses further prioritized variants with predicted functionality. Electrophoretic mobility shift assays (EMSAs) and luciferase expression were used to validate predicted SNPs in EBV transformed B (EBV B) cells.Results:While some differences were observed, the overall SS and SLE association signals were similar. SNP-SS rs9736016 nearCXCR5and SNP-SLE rs76409436 nearDDX6were the most significant but did not show evidence of functionality. Bayesian statistics defined credible sets of variants in strong D’ in common between both SS and SLE. Bioinformatics analyses (Haploreg, RegulomeDB, ENCODE data, etc) further refined the credible set and identified 5 common SNPs with strong evidence of functionality in immune cell types: rs4938572, rs4936443, rs57494551, rs7117261 and rs4938573. EMSAs showed a significant increase in protein binding to the risk allele of rs57494551 (p=0.0001), rs7117261 (p=0.0001) and rs4938573 (p=0.0003), but not the others, using nuclear lysates from EBV B cells. Luciferase vectors with a minimal promoter or no promoter were used to test for enhancer or promoter activity, respectively. To this end, the rs57494551 risk allele exhibited a significant increase in enhancer activity (p=0.0001). In contrast, the rs7117261 risk allele decreased enhancer activity (p=0.018). The rs4938573 risk allele decreased enhancer (p=0.043) and promoter (p=0.024) activity. While rs7117261 or rs4938573 were not reported in eQTL databases, GTex data reported rs57494551 as an eQTL that altersDDX6expression in whole blood (p=1.8E-7). Additionally, these functional SNPs have been associated with looping events to several proximal promoters in nearby genes in immune cells.Conclusion:SS and SLE have similar genomic architecture across theDDX6-CXCR5risk interval. Multiple variants in the credible set exhibited allele specific changes in protein binding, as well as modified enhancer activity, promoter activity or both. Ongoing studies will use Cas9 in EBV B cells to determine which other loci are within the local regulatory network.Disclosure of Interests:Mandi M Wiley: None declared, Bhuwan Khatri: None declared, Kandice L Tessneer: None declared, Michelle L Joachims: None declared, Anna M Stolarczyk: None declared, Astrid Rasmussen Speakers bureau: Novartis, ThermoFischer, Simon J. Bowman Consultant of: Astrazeneca, Biogen, BMS, Celgene, Medimmune, MTPharma, Novartis, Ono, UCB, xtlbio, Glapagos, Speakers bureau: Novartis, Lida Radfar: None declared, Roald Omdal: None declared, Marie Wahren-Herlenius: None declared, Blake M Warner: None declared, Torsten Witte: None declared, Roland Jonsson: None declared, Maureen Rischmueller: None declared, Patrick M Gaffney: None declared, Judith A. James Grant/research support from: Progentec Diagnostics, Inc, Consultant of: Abbvie, Novartis, Jannsen, Lars Ronnblom Grant/research support from: AZ, Speakers bureau: AZ, R Hal Scofield Grant/research support from: Pfizer, Xavier Mariette: None declared, Wan-fai Ng: None declared, Kathy L Sivils: None declared, Gunnel Nordmark: None declared, Betty Tsao: None declared, Christopher Lessard: None declared
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- Wiley, MM, et al.
(författare)
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SJOGREN'S DISEASE AND SYSTEMIC LUPUS ERYTHEMATOSUS DDX6-CXCR5 RISK INTERVALS REVEAL COMMON SNPS WITH FUNCTIONAL SIGNIFICANCE IN IMMUNE AND SALIVARY GLAND CELLS
- 2022
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Ingår i: ANNALS OF THE RHEUMATIC DISEASES. - : BMJ. - 0003-4967 .- 1468-2060. ; 81, s. 269-270
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- Sjögren’s Disease (SjD) and Systemic Lupus Erythematosus (SLE) are autoimmune diseases with several shared characteristics and similar genome-wide significant associations with the DDX6-CXCR5 locus. DDX6 suppresses interferon-stimulated gene expression and CXCR5 regulates T cell functions implicated in autoimmunity.ObjectivesTo identify and characterize functional SNPs in the DDX6-CXCR5 interval.MethodsImmunoChip data from European populations (3785 SLE cases; 1916 SjD cases; 6893 controls) were imputed and SNP-trait associations tested. Bayesian statistics defined a credible SNP set that was refined using bioinformatic analyses (RegulomeDB, Haploreg, ENCODE, promoter capture Hi-C, eQTLs, etc.). Electrophoretic mobility shift assays (EMSAs) and luciferase expression assays were used to test allele-specific SNP function in EBV-transformed B (EBV B) cells, Daudi B cells, Jurkat T cells, THP1 monocytes, and A253 salivary gland cell lines. Chromatin conformation capture with quantitative PCR (3C-qPCR) was used to assess long-range chromatin interactions.ResultsFine mapping of the SjD and SLE associations found similar SNP associations. Bioinformatic analyses identified 5 common SNPs with strong evidence of functionality in immune cell types: rs57494551 in an intron of DDX6, and rs4938572, rs4936443, rs7117261, and rs4938573 in the promoter/enhancer region of DDX6 and CXCR5. EMSAs and luciferase experiments showed cell type-specific differences in protein binding and promoter or enhancer activity, respectively, at each SNP. Risk allele of rs57494551 increased enhancer activity in B cells and A253 cells (p<0.001), but decreased promoter activity in T cells and A253 cells (p<0.01). SNP rs4938572 is an eQTL of DDX6 in T cells, and the risk allele significantly increased protein binding, promoter and enhancer activity in T cells (p<0.01). Risk allele of rs4938572 also increased promoter activity in A253 cells (p<0.001), but had no effect on promoter or enhancer activity in B cells. SNP rs4936443 showed no promoter or enhancer activity in immune cells, but the risk allele showed significant promoter and enhancer (p<0.001) activity in A253 cells. SNP rs7117261 showed decreased enhancer activity in EBV B cells, T cells, and A253 cells (p<0.05) and increased promoter activity in A253 cells (p<0.001). SNP rs4938573 showed decreased promoter activity in EBV B cells, T cell and A253 cells (p<0.05), decreased promoter activity in EBV B cells (p<0.05), and increased enhancer activity in A253 cells (p<0.0001). Overall, A253 cells exhibited more allele-specific effects on promoter and enhancer activity across the five SNPs compared to tested immune cells. In addition to DDX6 and CXCR5, rs57494551 and/or rs4938572 are reported eQTLs for several other genes of interest in the local chromatin regulatory network: IL10RA in T cells, TRAPPC4 in salivary gland and activated macrophages, and long non-coding (lnc)RNA AP002954.1 in T cells and whole blood. 3C-qPCR in EBV B and A253 cells showed that the two regulatory regions carrying rs4938572 or rs57494551 interacted with a region upstream of DDX6 that includes AP002954.1. Hi-C data showed looping between AP002954.1 and the regulatory region carrying rs4938572 and rs57494551 in T cells.ConclusionSjD and SLE share similar genomic architecture across the DDX6-CXCR5 risk interval with several common SNPs showing immune and salivary gland cell type-specific allelic effects on protein binding and/or enhancer/promoter activity. Extensive bioinformatic analyses suggest that the SNPs likely work within the local chromatin regulatory network to regulate cell type-specific expression of several genes on the interval. Ongoing studies will use 3C-qPCR to assess allele-specific chromatin interactions between the SNPs and these genes in different cells types, and CRISPR to determine how the risk alleles alters expression.Disclosure of InterestsMandi M Wiley: None declared, Bhuwan Khatri: None declared, Kandice L Tessneer: None declared, Michelle L Joachims: None declared, Anna M Stolarczyk: None declared, Anna Nagel: None declared, Astrid Rasmussen: None declared, Simon J. Bowman Consultant of: Abbvie, Galapagos, and Novartis in 2020-2021, Lida Radfar: None declared, Roald Omdal: None declared, Marie Wahren-Herlenius: None declared, Blake M Warner: None declared, Torsten Witte: None declared, Roland Jonsson: None declared, Maureen Rischmueller: None declared, Patrick M Gaffney: None declared, Judith A. James: None declared, Lars Ronnblom: None declared, R Hal Scofield: None declared, Xavier Mariette: None declared, Wan Fai Ng: None declared, Kathy Sivils Employee of: current employee of Janssen., Gunnel Nordmark: None declared, Betty Tsao: None declared, Christopher Lessard: None declared
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