Genetic variants and disease-associated factors contribute to enhanced interferon regulatory factor 5 expression in blood cells of patients with systemic lupus erythematosus.
ABSTRACT: Genetic variants of the interferon (IFN) regulatory factor 5 gene (IRF5) are associated with susceptibility to systemic lupus erythematosus (SLE). The contribution of these variants to IRF-5 expression in primary blood cells of SLE patients has not been addressed, nor has the role of type I IFNs. The aim of this study was to determine the association between increased IRF-5 expression and the IRF5 risk haplotype in SLE patients.IRF-5 transcript and protein levels in 44 Swedish patients with SLE and 16 healthy controls were measured by quantitative real-time polymerase chain reaction, minigene assay, and flow cytometry. Single-nucleotide polymorphisms rs2004640, rs10954213, and rs10488631 and the CGGGG insertion/deletion were genotyped in these patients. Genotypes of these polymorphisms defined both a common risk haplotype and a common protective haplotype.IRF-5 expression and alternative splicing were significantly up-regulated in SLE patients compared with healthy donors. Enhanced transcript and protein levels were associated with the risk haplotype of IRF5; rs10488631 displayed the only significant independent association that correlated with increased transcription from the noncoding first exon 1C. Minigene experiments demonstrated an important role for rs2004640 and the CGGGG insertion/deletion, along with type I IFNs, in regulating IRF5 expression.This study provides the first formal proof that IRF-5 expression and alternative splicing are significantly up-regulated in primary blood cells of patients with SLE. Furthermore, the risk haplotype is associated with enhanced IRF-5 transcript and protein expression in patients with SLE.
Project description:Systemic sclerosis (SSc) is a fibrotic autoimmune disease in which the genetic component plays an important role. One of the strongest SSc association signals outside the human leukocyte antigen (HLA) region corresponds to interferon (IFN) regulatory factor 5 (IRF5), a major regulator of the type I IFN pathway. In this study we aimed to evaluate whether three different haplotypic blocks within this locus, which have been shown to alter the protein function influencing systemic lupus erythematosus (SLE) susceptibility, are involved in SSc susceptibility and clinical phenotypes. For that purpose, we genotyped one representative single-nucleotide polymorphism (SNP) of each block (rs10488631, rs2004640, and rs4728142) in a total of 3,361 SSc patients and 4,012 unaffected controls of Caucasian origin from Spain, Germany, The Netherlands, Italy and United Kingdom. A meta-analysis of the allele frequencies was performed to analyse the overall effect of these IRF5 genetic variants on SSc. Allelic combination and dependency tests were also carried out. The three SNPs showed strong associations with the global disease (rs4728142: P ?=?1.34×10(-8), OR ?=?1.22, CI 95% ?=?1.14-1.30; rs2004640: P ?=?4.60×10(-7), OR ?=?0.84, CI 95% ?=?0.78-0.90; rs10488631: P ?=?7.53×10(-20), OR ?=?1.63, CI 95% ?=?1.47-1.81). However, the association of rs2004640 with SSc was not independent of rs4728142 (conditioned P ?=?0.598). The haplotype containing the risk alleles (rs4728142*A-rs2004640*T-rs10488631*C: P ?=?9.04×10(-22), OR ?=?1.75, CI 95% ?=?1.56-1.97) better explained the observed association (likelihood P-value ?=?1.48×10(-4)), suggesting an additive effect of the three haplotypic blocks. No statistical significance was observed in the comparisons amongst SSc patients with and without the main clinical characteristics. Our data clearly indicate that the SLE risk haplotype also influences SSc predisposition, and that this association is not sub-phenotype-specific.
Project description:OBJECTIVE: Interferon regulatory factor 5 (IRF-5) is a transcription factor involved in the regulation of the host defense. Previous studies have demonstrated a significant association of various IRF5 gene polymorphisms with systemic lupus erythematosus (SLE) in Caucasians. The purpose of this case-control study was to investigate whether IRF5 polymorphisms are involved in the genetic predisposition to primary Sjögren's syndrome (SS), an autoimmune disease closely related to SLE. METHODS: We analyzed IRF5 rs2004640, rs2070197, rs10954213, and rs2280714 polymorphisms in a cohort of 212 primary SS patients and 162 healthy blood donors, all of whom were of Caucasian origin. The 4 polymorphisms examined were genotyped by competitive allele-specific polymerase chain reaction using fluorescence resonance energy transfer technology. RESULTS: The IRF5 rs2004640 GT or TT genotype (T allele carriers) was identified in 87% of primary SS patients compared with 77% of controls (P = 0.01, odds ratio [OR] 1.93 [95% confidence interval (95% CI) 1.15-3.42]). The IRF5 rs2004640 T allele was found on 59% of chromosomes from primary SS patients compared with 52% of chromosomes from controls (P = 0.04, OR 1.36 [95% CI 1.01-1.83]). No significant association of primary SS with rs2070197, rs10954213, or rs2280714 was seen when they were analyzed independently. Nevertheless, haplotype reconstructions based on the 4 polymorphisms examined suggest that various allele combinations of rs2004640 and rs2070197 could define susceptibility or protective haplotypes. CONCLUSION: This study is the first to demonstrate a significant association between primary SS and the IRF5 rs2004640 T allele. These results, which require further replication on larger populations, suggest that besides their association with identical major histocompatibility complex gene polymorphisms, primary SS and SLE share IRF gene polymorphisms as a common genetic susceptibility factor.
Project description:A haplotype of the interferon regulatory factor 5 (IRF5) gene has been associated with the risk of developing systemic lupus erythematosus (SLE), and our previous studies have demonstrated that high levels of serum interferon-alpha (IFNalpha) activity are a heritable risk factor for SLE. The aim of this study was to determine whether the IRF5 SLE risk haplotype mediates the risk of SLE by predisposing patients to the development of high levels of serum IFNalpha activity.IFNalpha levels in 199 SLE patients of European and Hispanic ancestry were measured with a sensitive functional reporter cell assay. The rs2004640, rs3807306, rs10488631, and rs2280714 single-nucleotide polymorphisms (SNPs) in IRF5 were genotyped in these patients. Haplotypes were categorized as SLE risk, neutral, or protective based on published data.SLE patients with risk/risk and risk/neutral IRF5 genotypes had higher serum IFNalpha activity than did those with protective/protective and neutral/protective genotypes (P = 0.025). This differential effect of IRF5 genotype on serum IFNalpha levels was driven largely by SLE patients who were positive for either anti-RNA binding protein (anti-RBP) or anti-double-stranded DNA (anti-dsDNA) autoantibodies (P = 0.012 for risk/risk or risk/neutral versus protective/protective or neutral/protective). The rs3807306 genotype was independently associated with high serum IFNalpha in this autoantibody group. We found no difference in IFNalpha activity according to IRF5 genotype in patients lacking either type of autoantibody or in patients positive for both classes of autoantibody.The IRF5 SLE risk haplotype is associated with higher serum IFNalpha activity in SLE patients, and this effect is most prominent in patients positive for either anti-RBP or anti-dsDNA autoantibodies. This study demonstrates the biologic relevance of the SLE risk haplotype of IRF5 at the protein level.
Project description:Background & objectives:Genetic aberrations disrupting toll-like receptor and interferon homeostasis enhance the risk of systemic lupus erythematosus (SLE). Raised serum interferon-alpha (IFN-?) levels in SLE patients have been ascribed to polymorphism (rs2004640 G/T) in interferon regulatory factor 5 (IRF5) gene, resulting in enhanced transcript splicing. A positive association between IRF5 polymorphism and SLE risk has been reported in many populations. This study was aimed to find out frequency of IRF5 rs2004640 G/T polymorphism in patients with SLE and healthy controls and to assess its influence on susceptibility, clinical and serological characteristics of SLE. Methods:IRF5 rs2004640 (G/T) polymorphism was analyzed in 300 SLE patients and 460 age and sex matched controls by real-time PCR. Results:The IRF5 rs2004640 (G/T) polymorphism did not confer risk of SLE or influence clinical or serological phenotype. However, the mutant allele conferred a borderline risk to develop thrombocytopenia (odds ratio: 2.05, 95% confidence interval: 0.97-4.3, P=0.06) in patients with SLE. Interpretation & conclusions:Our study revealed that the IRF5 rs2004640 polymorphism was not a risk factor for SLE in population from south India. It may, however, be a useful genetic marker for thrombocytopenia in SLE patients. Although we could not demonstrate susceptibility toward lupus in the presence of IRF5 rs2004640 (G/T) polymorphism, further exploration of the genetic variability of IRF5 may help uncover its pathogenic role in Indian SLE patients.
Project description:Although several genetic associations with scleroderma (SSc) are defined, very little is known on genetic susceptibility to SSc-associated interstitial lung disease (SSc-ILD). A number of common polymorphisms have been associated with SSc-ILD, but most have not been replicated in separate populations. Four SNPs in IRF5, and one in each of STAT4, CD226 and IRAK1, selected as having been previously the most consistently associated with SSc-ILD, were genotyped in 612 SSc patients, of European descent, of whom 394 had ILD. The control population (n?=?503) comprised individuals of European descent from the 1000 Genomes Project. After Bonferroni correction, two of the IRF5 SNPs, rs2004640 (OR (95% CI)1.30 (1.10-1.54), pcorr?=?0.015) and rs10488631 (OR 1.48 (1.14-1.92), pcorr?=?0.022), and the STAT4 SNP rs7574865 (OR 1.43 (1.18-1.73), pcorr?=?0.0015) were significantly associated with SSc compared with controls. However, none of the SNPs were significantly different between patients with SSc-ILD and controls. Two SNPs in IRF5, rs10488631 (OR 1.72 (1.24-2.39), pcorr?=?0.0098), and rs2004640 (OR 1.39 (1.11-1.75), pcorr?=?0.03), showed a significant difference in allele frequency between controls and patients without ILD, as did STAT4 rs7574865 (OR 1.86 (1.45-2.38), pcorr?=?6.6?×?10-6). A significant difference between SSc with and without ILD was only observed for STAT4 rs7574865, being less frequent in patients with ILD (OR 0.66 (0.51-0.85), pcorr?=?0.0084). In conclusion, IRF5 rs2004640 and rs10488631, and STAT4 rs7574865 were significantly associated with SSc as a whole. Only STAT4 rs7574865 showed a significant difference in allele frequency in SSc-ILD, with the T allele being protective against ILD.Key points• We confirm the associations of the IRF5 SNPs rs2004640 and rs10488631, and the STAT4 SNP rs7574865, with SSc as a whole.• None of the tested SNPs were risk factors for SSc-ILD specifically.• The STAT4 rs7574865 T allele was protective against the development of lung fibrosis in SSc patients.• Further work is required to understand the genetic basis of lung fibrosis in association with scleroderma.
Project description:Results from two studies have implicated the interferon regulatory gene IRF5 as a susceptibility gene in systemic 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 = 3.4 x 10(-4)) and rs3807306 (P = 4.9 x 10(-4)), and the association extends into the 3'-untranslated region (UTR). There is a single haplotype block encompassing 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 = 1.92, P = 5.8 x 10(-5)), 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 3'-UTR. rs10954213 shows the strongest association with RNA expression levels (P = 1 x 10(-14)). 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 3'-UTR. Expression levels of transcript variants with the shorter or longer 3'-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.
Project description:The genetic association of interferon regulatory factor 5 (IRF5) with systemic lupus erythematosus (SLE) susceptibility has been convincingly established. To gain understanding of the effect of IRF5 variation in individuals without SLE, a study was undertaken to examine whether such genetic variation predisposes to activation of the interferon alpha (IFNalpha) pathway.Using a computer simulated approach, 14 single nucleotide polymorphisms (SNPs) and haplotypes of IRF5 were tested for association with mRNA expression levels of IRF5, IFNalpha and IFN-inducible genes and chemokines in lymphoblastoid cell lines (LCLs) from individuals of European (CEU), Han Chinese (CHB), Japanese (JPT) and Yoruba Nigerian (YRI) backgrounds. IFN-inducible gene expression was assessed in LCLs from children with SLE in the presence and absence of IFNalpha stimulation.The major alleles of IRF5 rs13242262 and rs2280714 were associated with increased IRF5 mRNA expression levels in the CEU, CHB+JPT and YRI samples. The minor allele of IRF5 rs10488631 was associated with increased IRF5, IFNalpha and IFN-inducible chemokine expression in CEU (p(c)=0.0005, 0.01 and 0.04, respectively). A haplotype containing these risk alleles of rs13242262, rs10488631 and rs2280714 was associated with increased IRF5, IFNalpha and IFN-inducible chemokine expression in CEU LCLs. In vitro studies showed specific activation of IFN-inducible genes in LCLs by IFNalpha.SNPs of IRF5 in healthy individuals of a number of ethnic groups were associated with increased mRNA expression of IRF5. In European-derived individuals, an IRF5 haplotype was associated with increased IRF5, IFNalpha and IFN-inducible chemokine expression. Identifying individuals genetically predisposed to increased IFN-inducible gene and chemokine expression may allow early detection of risk for SLE.
Project description:Polymorphisms in the interferon regulatory factor 5 (IRF5) gene have been consistently replicated and shown to confer risk for or protection from the development of systemic lupus erythematosus (SLE). IRF5 expression is significantly upregulated in SLE patients and upregulation associates with IRF5-SLE risk haplotypes. IRF5 alternative splicing has also been shown to be elevated in SLE patients. Given that human IRF5 exists as multiple alternatively spliced transcripts with distinct function(s), it is important to determine whether the IRF5 transcript profile expressed in healthy donor immune cells is different from that expressed in SLE patients. Moreover, it is not currently known whether an IRF5-SLE risk haplotype defines the profile of IRF5 transcripts expressed. Using standard molecular cloning techniques, we identified and isolated 14 new differentially spliced IRF5 transcript variants from purified monocytes of healthy donors and SLE patients to generate an IRF5 variant transcriptome. Next-generation sequencing was then used to perform in-depth and quantitative analysis of full-length IRF5 transcript expression in primary immune cells of SLE patients and healthy donors by next-generation sequencing. Evidence for additional alternatively spliced transcripts was obtained from de novo junction discovery. Data from these studies support the overall complexity of IRF5 alternative splicing in SLE. Results from next-generation sequencing correlated with cloning and gave similar abundance rankings in SLE patients thus supporting the use of this new technology for in-depth single gene transcript profiling. Results from this study provide the first proof that 1) SLE patients express an IRF5 transcript signature that is distinct from healthy donors, 2) an IRF5-SLE risk haplotype defines the top four most abundant IRF5 transcripts expressed in SLE patients, and 3) an IRF5 transcript signature enables clustering of SLE patients with the H2 risk haplotype.
Project description:Recently, two studies provided convincing evidence that IFN regulatory factor 5 (IRF5) gene polymorphisms are significantly associated with systemic lupus erythematosus (SLE) in several white populations. To replicate the association with SLE in an Asian population, we examined the genetic effects in our SLE cohort from a Korean population. A total of 1,565 subjects, composed of 593 cases and 972 controls, were genotyped using the TaqMan (Applied Biosystems, Foster City, CA, USA) method. The genetic effects of polymorphisms on the risk of SLE were evaluated using chi2 tests and a Mantel-Haenszel meta-analysis. Statistical analysis revealed results in the Korean population were similar to the previous reports from white populations. The rs2004640 T allele had a higher frequency in SLE cases (0.385) than controls (0.321; odds ratio (OR) = 1.32, P = 0.0003). In combined analysis, including all seven independent cohorts from the three studies so far, robust and consistent associations of the rs2004640 T allele with SLE were observed. The estimate of risk was OR = 1.44 (range, 1.34-1.55), with an overall P = 1.85 x 10(-23) for the rs2004640 T allele. The haplotype (rs2004640T-rs2280714T) involved in both the alternative splice donor site and the elevated expression of IRF5 also had a highly significant association with SLE (pooled, P = 2.11 x 10(-16)). Our results indicate that the genetic effect on the risk of SLE mediated by IRF5 variants can be generally accepted in both white and Asian populations.
Project description:BACKGROUND: Recently, a new genetic factor within the interferon regulatory factor 5 (IRF5) gene was demonstrated for systemic lupus erythematosus (SLE) through linkage and association: the rs2004640-T allele. IRF5 is involved in the production of rheumatoid arthritis (RA) cytokines, and SLE already shares with RA one genetic factor within the tyrosine phosphatase PTPN22 gene. AIM: To test the hypothesis that the SLE IRF5 genetic factor could also be shared with RA. PATIENTS AND METHODS: 100 French Caucasian trio families with RA were genotyped and analysed with the transmission disequilibrium test, the frequency comparison of the transmitted and untransmitted alleles, and the genotype relative risk. 97% power was available to detect at least a trend in favour of a factor similar to that reported for SLE. RESULTS: The analysis showed the absence of linkage and association globally and in "autoimmune" RA subsets, with a weak non-significant trend against the IRF5 rs20046470-T allele. Given the robustness of familial-based analysis, this slight negative trend provided strong evidence against even a weaker factor than that reported for SLE. CONCLUSION: Our results exclude the IRF5 rs2004640-T allele as a major genetic factor for RA in this French Caucasian population.