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Keywords:

  • interferon regulatory factor 5;
  • IRF5;
  • interferon;
  • lupus;
  • SLE

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Subjects and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Interferon regulatory factor 5 (IRF5) belongs to a family of transcription factors that control the transactivation of type I interferon system-related genes, as well as the expression of several other genes involved in immune response, cell signalling, cell cycle control and apoptosis. Two recent studies reported a significant association between the IRF5/rs2004640 T allele and systemic lupus erythematosus (SLE). The purpose of this study was to determine whether the reported rs2004640 T allele association could be replicated in our independent SLE case-control sample. We genotyped DNA samples from 370 white SLE-affected female subjects and 462 white healthy female controls using the TaqMan Assay-on-Demand for rs2004640, and performed a case-control genetic association analysis. Frequency of the rs2004640 T allele was significantly higher in cases than in controls (56.5% vs. 50%; P= 0.008). The odds ratio for T allele carriers was 1.68 (95% CI: 1.20 – 2.34; P= 0.003). Our results in an independent case-control sample confirm the robust association of the IRF5/rs2004640 T allele with SLE risk, and further support the relevance of the type I interferon system in the pathogenesis of SLE and autoimmunity.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Subjects and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Systemic lupus erythematosus (SLE) is a prototypic multi-system autoimmune disease that predominantly affects premenopausal women. The disease is characterized by systemic chronic inflammation associated with the production of autoantibodies against multiple antigens, including nucleic acids and nucleoproteins. SLE has a complex genetic basis and is caused by the complex interaction of unknown environmental factors and multiple genetic susceptibility loci on different chromosomes (Nath et al. 2004).

Several studies have suggested that the type I interferon (IFN) system plays a central role in the initiation and progression of SLE (Crow, 2005; Dall'era et al. 2005). Elevated serum IFN-alpha levels and increased expression of type I IFN-inducible genes have been shown to be correlated with SLE activity and severity, as well as with several markers of immune activation (anti-dsDNA titers, presence of anti-RNA binding protein autoantibodies, degree of complement activation) (Crow, 2005; Dall'era et al. 2005). The long-term therapeutic use of IFN-alpha can occasionally induce SLE manifestations, and trisomy of the type I interferon cluster on chromosome 9p was reported to be associated with SLE-like disease, further supporting the critical role of the type I IFN system in the etiopathogenesis of SLE (Zhuang et al. 2006). IFN regulatory factor 5 (IRF5) belongs to a family of transcription factors that control the transactivation of the type I IFN system-related genes, as well as the expression of several other genes involved in immune response, cell signalling, cell cycle control and apoptosis (Barnes et al. 2003, 2004; Izaguirre et al. 2003; Coccia et al. 2004; Mancl et al. 2005). The function of a particular IRF is determined by its cell type-specific expression, its intrinsic transactivation potential, and its interactions with other members of the IRF family, other transcription factors, and members of the TLR signalling pathway (Barnes et al. 2003, 2004; Izaguirre et al. 2003; Coccia et al. 2004; Mancl et al. 2005; Schoenemeyer et al. 2005; Takaoka et al. 2005). Multiple IRF5 splice isoforms have been identified and encode proteins with distinct cell type-specific expression, cellular localization, differential regulation, and dissimilar functions in type I IFN gene induction (Mancl et al. 2005).

Recently, two studies reported significant association between the IRF5/rs2004640 T allele and SLE (Sigurdsson et al. 2005; Graham et al. 2006). The T allele creates a 5′ donor splice site for an alternative exon (exon 1B) of IRF5, allowing the expression of transcripts containing this alternate exon (Graham et al. 2006). Since replications in independent cohorts by different groups are imperative for establishing genetic associations, we sought to replicate the previously reported association between the rs2004640 T allele and SLE risk in our independent case-control cohort.

Subjects and Methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Subjects and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Subjects

Peripheral blood leukocyte DNA samples from 370 white SLE women (308 from Pittsburgh and 62 from Chicago) and 462 white control women (407 from Pittsburgh and 55 from Chicago) were used in the present study.

The SLE cases were recruited for a multi-centre study designed to determine the prevalence of cardiovascular disease and associated risk factors in women with SLE. Cases were 18 years of age or older, and met the 1982 and 1997 revised American College of Rheumatology classification criteria for SLE (Hochberg, 1997). All SLE subjects were participants in either the Pittsburgh Lupus Registry or the Chicago SOLVABLE study (Study Of Lupus Vascular And Bone Longterm Endpoints). Demographic and clinical details of the patient population have been described elsewhere (Selzer et al. 2001; Tripi et al. 2006). Controls with no apparent history of SLE were geographically matched and obtained either from the Central Blood Bank of Pittsburgh or from the SOLVABLE study for the Chicago site.

Blood samples were obtained at the baseline visit. Informed consent was obtained prior to participation to this study, in accordance with protocols approved by the University of Pittsburgh and the Northwestern University Institutional Review Boards.

Genotyping

IRF5 SNP (rs2004640) genotyping was performed using a pre-made TaqMan® SNP Genotyping Assay (C__9491614_10) (Applied Biosystems, Foster City, CA) for TaqMan® allelic discrimination. The end-point fluorescence readings were performed with an ABI Prism 7900 HT Sequence Detection System (Applied Biosystems) using 384-well plates.

Statistical Methods

Allele frequencies were calculated by the allele counting method. Observed genotype frequencies were compared to Hardy-Weinberg equilibrium, and the significance of deviations was tested by the chi-squared goodness-of-fit test. Pearson's chi-squared test and standard Z-test of two binomial proportions were used to compare the genotype and allele frequencies, respectively, between cases and controls. All computations and regression analyses were performed using the R statistical software package (version 2.1.1, http://www.r-project.org).

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Subjects and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Genotype frequencies were found to be in Hardy-Weinberg equilibrium in both the Pittsburgh and Chicago groups for both cases and controls. The genotype distributions and allele frequencies showed significant differences between SLE patients and healthy controls in the Pittsburgh sample (P= 0.037 and P= 0.028, respectively) and in the combined Pittsburgh+Chicago sample (P= 0.009 and P= 0.008, respectively) (Table 1). Despite a similar trend also being observed in the Chicago group, the sample size was not large enough to yield statistically significant results. The rs2004640 T allele frequency was 55.8% in SLE cases vs. 50% in controls in the Pittsburgh sample, 59.7%vs. 50% in the Chicago sample, and 56.5%vs. 50% in the combined Pittsburgh+Chicago sample. The OR for the T allele carriers (TT+TG vs GG genotypes) was 1.60 (95% CI: 1.11 – 2.29; P= 0.011) in the Pittsburgh sample, 2.21 (95% CI: 0.88 – 5.56; P= 0.092) in the Chicago sample, and 1.68 (95% CI: 1.20 – 2.34; P= 0.003) in the combined Pittsburgh+Chicago sample.

Table 1.  Frequency of IRF5/rs2004640 SNP in SLE patients and controls
Population GroupPatientsControlsOdds ratios for genotypes
Pittsburgh(n= 308)(n= 407) 
Genotypes n (%) n (%) OR (95% CI)
TT 94 (30.52)110 (27.03)1.62 (1.06-2.47)
TG 156 (50.65)187 (45.95)1.58 (1.08-2.32)
GG 58 (18.83)110 (27.03)-
P= 0.037
Alleles
T 0.5580.500 
G 0.4420.500 
P= 0.028
Chicago(n= 62)(n= 55) 
Genotypes n (%) n (%) OR (95% CI)
TT 21 (33.87)15 (27.27)2.33 (0.81-6.73)
TG 32 (51.61)25 (45.45)2.13 (0.80-5.67)
GG 9 (14.52)15 (27.27) - 
P= 0.229
Alleles
T 0.5970.500 
G 0.4030.500 
P= 0.136
Combined(n= 370)(n= 462) 
Genotypes n (%) n (%) OR (95% CI)
TT 115 (31.08)125 (27.06)1.72 (1.16-2.54)
TG 188 (50.81)212 (45.89)1.65 (1.16-2.36)
GG 67 (18.11)125 (27.06) - 
P= 0.009
Alleles
T 0.5650.500 
G 0.4350.500 
P= 0.008

Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Subjects and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

IRF5 is known to regulate type I IFN production and the induction of various proinflammatory cytokines (such as interleukin-6, interleukin-12, and tumor necrosis factor-alpha) (Barnes et al. 2003, 2004; Izaguirre et al. 2003; Coccia et al. 2004; Mancl et al. 2005). It has been proposed to act as both an activator and a repressor of IFN gene induction, depending on the IRF-interacting partner, and is considered to be part of the regulatory network that controls timely expression of the early inflammatory genes. Therefore, a functional IRF5 genetic variant may affect several immune functions that are important for the development and progression of autoimmune diseases such as SLE.

Two recent studies have reported an association between the IRF5/rs2004640 SNP and SLE (Sigurdsson et al. 2005; Graham et al. 2006). Initially, Sigurdsson et al. (2005) analyzed 44 SNPs in 13 genes from the type I IFN pathway in Swedish and Finnish cohorts, and found significant association with the IRF5/rs2004640 SNP in both cohorts. Subsequently, Graham et al. (2006) replicated the association of the IRF5/rs2004640 T allele with SLE in four independent cohorts (from Argentina, Spain, Sweden, and USA) using case-control association and family-based transmission disequilibrium test analyses. The rs2004640 T allele frequency was 61% in cases and 51% in controls in the American SLE case-control sample reported by the latter study (Graham et al. 2006). These frequencies were 56.5% and 50%, respectively, in our combined USA sample. The T allele-associated ORs in the six reported cohorts varied between 1.31 and 1.84, with a pooled OR of 1.47 (95% CI: 1.36-1.60) which is similar to our genotype-derived (TT+TG vs GG) OR of 1.68. These data provide compelling evidence that this is a genuine association with a modest effect. Interestingly the IRF5 gene is located on chromosome 7q32 which is not among the SLE linkage regions that have been reported to date. This reinforces the concept that association studies are more powerful than linkage studies to detect modest effect sizes.

In conclusion, our data in conjunction with the previously published data indicate that the IRF5/rs2004640 T allele is a genuine risk marker for SLE. Since the scope of this study was to replicate the reported association of the IRF5/rs2004640 SNP in an independent Caucasian sample we did not attempt to analyze additional SNPs in this gene. Our replication data provide a strong justification for a comprehensive examination of the IRF5 gene to detect additional common and/or rare variants that affect SLE risk. Understanding the molecular mechanisms pertaining to IRF5 function and the spectrum of the IRF5-targeted genes will increase our knowledge about SLE ethiopathogenesis and may guide novel therapeutic strategies for SLE and other autoimmune diseases.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Subjects and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

We are grateful to the subjects who participated in this study. This study was supported by the National Heart, Lung, and Blood Institute Grants HL 074165 and HL54900, the National Institute of Arthritis and Musculoskeletal and Skin Diseases Grants AR 46588, AR 002213, AR 02318, AR 48098, F32 AR 51681, and the grants from the NIH General Clinical Research Center M01-RR000056 and M01-RR000048.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Subjects and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
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