The molecular genetic basis of the association of TNFSF4 with SLE

The tumour necrosis factor ligand superfamily member 4 gene (TNFSF4), also known as OX40L, is an established susceptibility locus in the autoimmune disease systemic lupus erythematosus (SLE). Genetic association studies map polymorphisms that associate with disease, but linkage disequilibrium often hinders the identification of the actual casual allele(s) at a disease susceptibility locus. At TNFSF4 genetic association studies had shown that an extended 100kb haplotype upstream of the coding region of the gene was associated with SLE risk. The principle aim of the project was to conduct genetic association analyses in cohorts with different ancestry in an attempt to fine map the TNFSF4 association signal and thereby identify the causal genetic variants that underlie the genetic risk. Utilizing >17,900 subjects of European, African-American, Hispanic-American and Southeast Asian ancestry a transancestral fine mapping analysis was performed. The results demonstrate the strong association of TNFSF4 risk alleles in all populations tested. The most consistent and strongest evidence of association came from the single nucleotide polymorphism (SNP), rs2205960-T (P = 7.1 x 10-32, odds ratio = 1.63). This variant was also associated with autoantibody production in three independent cohorts. In silico analysis of the DNA sequence encompassing rs2205960-T predicts it to form part of a decameric motif, which binds the RelA (p65) component of the NF-κB transcription factor complex. A second associated SNP, rs16845607-A in TNFSF4 intron 1 was identified in Hispanic-Americans (P = 9.17 x 10-9, odds ratio = 2.06). In an attempt to further refine the association, resequencing was performed in 80 individuals who were selected on the basis of their genotype to carry risk or non-risk haplotypes upstream of TNFSF4. This sequencing study identified >200 novel variants, mostly small insertion-deletion polymorphisms indels. The data presented in this thesis largely resolves the genetic basis of the immediate upstream association signal observed at TNFSF4 with SLE and will facilitate the unraveling of the molecular basis of this genetic risk in systemic autoimmunity.Open Acces

Trans-Ancestral Studies Fine Map the SLE-Susceptibility Locus TNFSF4

We previously established an 80 kb haplotype upstream of TNFSF4 as a susceptibility locus in the autoimmune disease SLE. SLE-associated alleles at this locus are associated with inflammatory disorders, including atherosclerosis and ischaemic stroke. In Europeans, the TNFSF4 causal variants have remained elusive due to strong linkage disequilibrium exhibited by alleles spanning the region. Using a trans-ancestral approach to fine-map the locus, utilising 17,900 SLE and control subjects including Amerindian/Hispanics (1348 cases, 717 controls), African-Americans (AA) (1529, 2048) and better powered cohorts of Europeans and East Asians, we find strong association of risk alleles in all ethnicities; the AA association replicates in African-American Gullah (152,122). The best evidence of association comes from two adjacent markers: rs2205960-T (P = 1.71×10-34, OR = 1.43[1.26-1.60]) and rs1234317-T (P = 1.16×10-28, OR = 1.38[1.24-1.54]). Inference of fine-scale recombination rates for all populations tested finds the 80 kb risk and non-risk haplotypes in all except African-Americans. In this population the decay of recombination equates to an 11 kb risk haplotype, anchored in the 5′ region proximal to TNFSF4 and tagged by rs2205960-T after 1000 Genomes phase 1 (v3) imputation. Conditional regression analyses delineate the 5′ risk signal to rs2205960-T and the independent non-risk signal to rs1234314-C. Our case-only and SLE-control cohorts demonstrate robust association of rs2205960-T with autoantibody production. The rs2205960-T is predicted to form part of a decameric motif which binds NF-κBp65 with increased affinity compared to rs2205960-G. ChIP-seq data also indicate NF-κB interaction with the DNA sequence at this position in LCL cells. Our research suggests association of rs2205960-T with SLE across multiple groups and an independent non-risk signal at rs1234314-C. rs2205960-T is associated with autoantibody production and lymphopenia. Our data confirm a global signal at TNFSF4 and a role for the expressed product at multiple stages of lymphocyte dysregulation during SLE pathogenesis. We confirm the validity of trans-ancestral mapping in a complex trait. © 2013 Manku et al

The Innate Immune Responses of Colonic Epithelial Cells to Trichuris muris Are Similar in Mouse Strains That Develop a Type 1 or Type 2 Adaptive Immune Response

Trichuris muris resides in intimate contact with its host, burrowing within cecal epithelial cells. However, whether the enterocyte itself responds innately to T. muris is unknown. This study investigated for the first time whether colonic intestinal epithelial cells (IEC) produce cytokines or chemokines following T. muris infection and whether divergence of the innate response could explain differentially polarized adaptive immune responses in resistant and susceptible mice. Increased expression of mRNA for the proinflammatory cytokines gamma interferon (IFN-γ) and tumor necrosis factor and the chemokine CCL2 (MCP-1) were seen after infection of susceptible and resistant strains, with the only difference in expression being a delayed increase in CCL2 in BALB/c IEC. These increases were ablated in MyD88(−/−) mice, and NF-κB p65 was phosphorylated in response to T. muris excretory/secretory products in the epithelial cell line CMT-93, suggesting involvement of the MyD88-NF-κB signaling pathway in IEC cytokine expression. These data reveal that IEC respond innately to T. muris. However, the minor differences identified between resistant and susceptible mice are unlikely to underlie the subsequent development of a susceptible type 1 (IFN-γ-dominated) or resistant type 2 (interleukin-4 [IL-4]/IL-13-dominated) adaptive immune response

Association of IRF5 in UK SLE families identifies a variant involved in polyadenylation

Results from two studies have implicated the interferon regulatory gene IRF5 as a susceptibility gene in sys-temic lupus erythematosus (SLE). In this study, we conducted a family-based association analysis in 380 UK SLE nuclear families. Using a higher density of markers than has hitherto been screened, we show that there is association with two SNPs in the first intron, rs2004640 (P 5 3.4 3 1024) and rs3807306 (P 5 4.9 3 1024), and the association extends into the 30-untranslated region (UTR). There is a single haplotype block encom-passing IRF5 and we show for the first time that the gene comprises two over-transmitted haplotypes and a single under-transmitted haplotype. The strongest association is with a TCTAACT haplotype (T:U 5 1.92, P 5 5.8 3 1025), which carries all the over-transmitted alleles from this study. Haplotypes carrying the T alleles of rs2004640 and rs2280714 and the A allele of rs10954213 are over-transmitted in SLE families. The TAT haplotype shows a dose-dependent relationship with mRNA expression. A differential expression pattern was seen between two expression probes located each side of rs10954213 in the 30-UTR. rs10954213 shows the strongest association with RNA expression levels (P 5 1 3 10214). The A allele of rs10954213 creates a functional polyadenylation site and the A genotype correlates with increased expression of a transcript variant containing a shorter 30-UTR. Expression levels of transcript variants with the shorter or longer 30-UTRs are inversely correlated. Our data support a new mechanism by which an IRF5 polymorphism controls the expression of alternate transcript variants which may have different effects on interferon signalling

Expansion of Circulating T Cells Resembling Follicular Helper T Cells Is a Fixed Phenotype That Identifies a Subset of Severe Systemic Lupus Erythematosus

Objective. In the sanroque mouse model of lupus, pathologic germinal centers (GCs) arise due to increased numbers of follicular helper T (Tfh) cells, resulting in high-affinity anti-double-stranded DNA antibodies that cause end-organ inflammation, such as glomerulonephritis. The purpose of this study was to examine the hypothesis that this pathway could account for a subset of patients with systemic lupus erythematosus (SLE). Methods. An expansion of Tfh cells is a causal, and therefore consistent, component of the sanroque mouse phenotype. We validated the enumeration of circulating T cells resembling Tfh cells as a biomarker of this expansion in sanroque mice, and we performed a comprehensive comparison of the surface phenotype of circulating and tonsillar Tfh cells in humans. This circulating biomarker was enumerated in SLE patients (n = 46), Sjögren's syndrome patients (n = 17), and healthy controls (n = 48) and was correlated with disease activity and end-organ involvement. Results. In sanroque mice, circulating Tfh cells increased in proportion to their GC counterparts, making circulating Tfh cells a feasible human biomarker of this novel mechanism of breakdown in GC tolerance. In a subset of SLE patients (14 of 46), but in none of the controls, the levels of circulating Tfh cells (defined as circulating CXCR5+CD4+ cells with high expression of Tfh-associated molecules, such as inducible T cell co-stimulator or programmed death 1) were increased. This cellular phenotype did not vary with time, disease activity, or treatment, but it did correlate with the diversity and titers of autoantibodies and with the severity of end-organ involvement. Conclusion. These findings in SLE patients are consistent with the autoimmune mechanism in sanroque mice and identify Tfh effector molecules as possible therapeutic targets in a recognizable subset of patients with SLE

Polymorphism at the TNF superfamily gene TNFSF4 confers susceptibility to systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is a multisystem complex autoimmune disease of uncertain etiology (OMIM 152700). Over recent years a genetic component to SLE susceptibility has been established. Recent successes with association studies in SLE have identified genes including IRF5 (refs. 4,5) and FCGR3B. Two tumor necrosis factor (TNF) superfamily members located within intervals showing genetic linkage with SLE are TNFSF4 (also known as OX40L; 1q25), which is expressed on activated antigen-presenting cells (APCs) and vascular endothelial cells, and also its unique receptor, TNFRSF4 (also known as OX40; 1p36), which is primarily expressed on activated CD4+ T cells. TNFSF4 produces a potent co-stimulatory signal for activated CD4+ T cells after engagement of TNFRSF4 (ref. 11). Using both a family-based and a case-control study design, we show that the upstream region of TNFSF4 contains a single risk haplotype for SLE, which is correlated with increased expression of both cell-surface TNFSF4 and the TNFSF4 transcript. We hypothesize that increased expression of TNFSF4 predisposes to SLE either by quantitatively augmenting T cell-APC interaction or by influencing the functional consequences of T cell activation via TNFRSF4

Trans-Ancestral Studies Fine Map the SLE-Susceptibility Locus TNFSF4

We previously established an 80 kb haplotype upstream of TNFSF4 as a susceptibility locus in the autoimmune disease SLE. SLE-associated alleles at this locus are associated with inflammatory disorders, including atherosclerosis and ischaemic stroke. In Europeans, the TNFSF4 causal variants have remained elusive due to strong linkage disequilibrium exhibited by alleles spanning the region. Using a trans-ancestral approach to fine-map the locus, utilising 17,900 SLE and control subjects including Amerindian/Hispanics (1348 cases, 717 controls), African-Americans (AA) (1529, 2048) and better powered cohorts of Europeans and East Asians, we find strong association of risk alleles in all ethnicities; the AA association replicates in African-American Gullah (152,122). The best evidence of association comes from two adjacent markers: rs2205960-T (P = 1.71×10-34, OR = 1.43[1.26-1.60]) and rs1234317-T (P = 1.16×10-28, OR = 1.38[1.24-1.54]). Inference of fine-scale recombination rates for all populations tested finds the 80 kb risk and non-risk haplotypes in all except African-Americans. In this population the decay of recombination equates to an 11 kb risk haplotype, anchored in the 5′ region proximal to TNFSF4 and tagged by rs2205960-T after 1000 Genomes phase 1 (v3) imputation. Conditional regression analyses delineate the 5′ risk signal to rs2205960-T and the independent non-risk signal to rs1234314-C. Our case-only and SLE-control cohorts demonstrate robust association of rs2205960-T with autoantibody production. The rs2205960-T is predicted to form part of a decameric motif which binds NF-κBp65 with increased affinity compared to rs2205960-G. ChIP-seq data also indicate NF-κB interaction with the DNA sequence at this position in LCL cells. Our research suggests association of rs2205960-T with SLE across multiple groups and an independent non-risk signal at rs1234314-C. rs2205960-T is associated with autoantibody production and lymphopenia. Our data confirm a global signal at TNFSF4 and a role for the expressed product at multiple stages of lymphocyte dysregulation during SLE pathogenesis. We confirm the validity of trans-ancestral mapping in a complex trait. © 2013 Manku et al

Conditional regression results for 5′TNFSF4 variants in four groups.

<p>Conditional analyses in SNPTESTv2 Case Control. Continuous covariate within a clustering framework. P values selected using additional model and a frequentist paradigm.</p

LD plots at <i>TNFSF4</i> locus in four populations.

<p>This section of chromosome 1q25.1 encompasses the <i>TNFSF4</i> gene and upstream region as defined by custom algorithm in Haploview 4.2. The measure of LD was used to depict 57 SNPs common to all cohorts, post QC and 1000 genomes imputation. The pair-wise correlations between <i>TNFSF4</i> markers is illustrated in these plots by the correlation coefficient R²(where r² = 0 = no correlation, white; 02<1, gradations of grey; R² = 1 = complete correlation, black). The <i>TNFSF4</i> gene is positioned above the plots relative to haplotype blocks (black triangles) and grey ticks indicate SNP locations to scale.</p