We have identified robust associations between the STAT4 (but not STAT1) gene and multiple SNPs in a large study of SLE cases and controls from different racial backgrounds (best combined p=7.02×10−25). To our knowledge this is the first study that targets both STAT4 and STAT1 genes with high resolution SNP genotyping in an extensive case-control study that includes high risk minority African-American, Hispanic, and Korean-Asian populations and a large cohort of childhood-onset SLE.
We found that the observed association with STAT1 is orders of magnitude weaker than that of STAT4 with respect to both p values and number of significant SNPs. This suggestive association with STAT1 most likely reflects the LD with STAT4 since STAT1 and STAT4 are only 25 kb apart. Indeed, we observed (D′=0.96) between rs10199181 in STAT1 among Europeans which produced the best suggestive result (p =1.40 ×10−3) in this gene and the nearest marker on 3′ end of STAT4 gene rs3024896 (p=6.13×10−6). However, this suggestive result disappeared completely p=0.60 upon conditional analyses. Although the genetic association between SLE and STAT1 cannot be formally excluded, our collection of almost 10,000 samples, which has a 99% power to find effects with odds ratio (OR) = 1.3 at p=10−8, and 84% power to find effects with OR =1.2 at p=10−6 for SNPs with a minor allele frequency of 0.3 and D′=1, makes it highly unlikely that STAT1 is a susceptibility gene for SLE.
Remmers et al
) have demonstrated association of one STAT4 SNP (rs7574865) with SLE in Europeans. Although we did not directly type rs7574865 in our samples, the HapMap CEU data indicate that rs7574865, is a perfect proxy of rs7568275 in European Americans (r2
=1). The SNP rs7568275 was one of the top associated SNPs in our data with Fisher combined p= 1.08×10−22
( and supplementary Table 1
). Using HapMap CEU data in European, imputation method support this proxy association for SNP rs7574865 with p=6.41×10−15
in our data. Both of these SNPs are located in the third intron of STAT4.
Lee et al
) replicated the association of STAT4 with RA in European and Korean patients. Three SNPs (rs10181656, rs13017460, and rs1517352) that were significantly associated with rheumatoid arthritis in Korean patients, were also significant in both European and Asian cases in our SLE subjects with the same associated alleles ( and supplementary Table 1
). In addition, the minor allele frequencies observed between our study and the studies by Remmers et al
. and Lee at al
. are similar.
Also, Korman et al
) reported an association with rs7574865 and primary Sjogren’s syndrome (PSS) in a study of 124 Caucasian PSS subjects and 1143 controls (p=0.01). PSS and SLE share overlapping autoantibody profiles (such as anti-Ro) and B lymphocyte hyperactivity, supporting the notion that related autoimmune diseases share common risk variants in STAT4.
Using a relatively dense map of SNPs within the STAT4 gene enabled us to construct haplotypes in the various racial groups. While we identified and strongly confirmed multiple SNP associations in 3rd
intron of STAT4 gene, we also detected two additional haplotypes adjacent to this intron by dense SNP genotyping that spans from exon 4 to exon 17 of this gene. Most importantly, one of these haplotypes, an eight marker haplotype spanning 18 kb from exon 4 to exon 14 of the STAT4 gene was significant in multiple ethnicities. Conditional analyses on this haplotype suggest that rs10168266 can explain the whole association in this haplotype and, therefore, the genotyping data for this SNP can predict the risk or protective haplotype. Indeed, this SNP produced the best results in European, Korean-Asian, and Hispanic with the combined Fisher p value of p= 1.12×10−24
in our study ( and supplementary Table 1
). The best model of association for this SNP (and most other highly significant markers) was an additive model (European p= 7.80×10−16
) (supplementary Table 1
). This intronic SNP is located between exons 5 and 6 of STAT4 gene and is 28 kb apart from the published rs7574865 at 3rd
intron with the estimated LD=0.90 and r2
=0.62 between them in European population. However as mentioned previously they are located on different but adjacent haplotypes (block 2 and block 3 respectively) ().
Functionally, STAT 4 is the main transcriptional regulatory molecule for IL-12 and, as such, is pivotal to the development of a fully functioning Th1 immune response (38
). Polarization of the immune response to Th1 vs Th2 has profound in vivo consequences. In general,
Th1-type immune res ponses are characteristic of cell-mediated immunity, while Th2-type responses are associated with help for B-cell antibody production (humoral immunity) and allergic phenomena (39
). Indeed, STAT4-deficient mice are protected from the effects of Th1-cell mediated autoimmune diseases. In models of experimental allergic encephalomyelitis (EAE) (40
), experimental arthritis (41
), colitis (42
), myocarditis (43
), and diabetes in the NOD mouse (44
), STAT4-deficient mice display less disease, decreased parameters of inflammation, and reduced secretion of IFNγ. Although IFNγ-deficiency does mimic STAT4 deficiency in an arthritis model (41
), IFNγ-deficient mice are not protected from EAE (45
), myocarditis (43
), or colitis (42
). Thus, although IFNγ is an important STAT4-induced immune mediator, STAT4 regulates other genes independent of IFNγ that are crucial for the development of such diseases. By contrast, in other autoimmune diseases that are not Th1 mediated, STAT4-deficient mice are not protected from autoimmune diseases that are not Th1-mediated, such as myasthenia gravis or Graves’ disease (46
). Moreover, STAT4-deficiency caused more severe SLE-like disease in NZM 2328 and NZM 2410 mice than in corresponding wild-type control mice (48
Since other cytokines (IL-23, IFNα) in addition to IL-12, can activate STAT4 (20
), the phenotype of STAT4-deficient mice, in the different mouse models, may actually be a composite effect of defects in IL-12, IL-23, and IFNα signaling, in addition to other yet unidentified cytokine pathways (20
). It should also be stressed that the different effects of STAT4 deficiency in animal models of arthritis compared to those in SLE point to the real possibility that the causal STAT4 polymorphisms in SLE and RA differ from each other.
A relatively small replication study in Swedish SLE patients suggest a significant correlation between the European risk allele and production of anti-dsDNA autoantibodies (50
) while a second study in North Americans of European ancestry suggest a strong correlation between SNP rs7574865 and anti-dsDNA Abs and an even stronger association with nephritis (51
). These subphenotype associations should be interpreted with caution given that the same SNP alleles are associated with RA patients that do not have anti-dsDNA autoAbs and do not develop nephritis. In this regards, the in vivo
findings that STAT4 deficient lupus mice develop accelerated nephritis despite decreased levels of anti-dsDNA autoAbs (48
) might be highly relevant. Subphenotype analyses of our data, in a much larger collection of SLE subjects then these previous studies, do not support a stronger association between STAT4 risk alleles and autoAb levels or presence of nephritis. For example, rs1068266, that produces the best association with nephritis in our European cases (OR=1.59) was not statistically significant from the results obtained with the same SNP in Europeans without nephritis (OR=1.46).
Based on the nature of the SNPs implicated in the present study, it is premature to suggest a molecular mechanism that would explain the gene’s association with SLE. Although we used a relatively dense SNP map, the possibility of other polymorphisms that may be responsible for the exact disease mechanism still exists and must await a complete resequencing of the STAT4 gene in SLE patients. Nevertheless, our findings here should significantly advance our understanding and establish new key steps in the pathogenesis of the disease. Furthermore, the unambiguous establishment of STAT4 as a susceptibility gene for SLE provides justification for the developments of therapeutic approaches targeting this molecule or other molecules within its biochemical pathway.