The STAT4 gene is located on 2q32.2-q32.3 adjacently to STAT1 gene, and the region encompassing STAT1 and STAT4 spans approximately 180 kilobase pairs. In the first screening, 52 tag SNPs in the STAT1-STAT4 region, selected with an r2 threshold of 0.9 based on the HapMap Phase II JPT data, were genotyped in 105 Japanese female SLE patients and 102 female healthy controls, and allele frequencies were compared between SLE patients and controls. A linkage disequilibrium (LD) plot and the results of the association study in the STAT1-STAT4 region are shown in Figure . Pairwise r2 values between 52 tag SNPs were calculated using genotyping data from 102 healthy individuals.
Figure 1 Linkage disequilibrium plot of the STAT1-STAT4 region in a Japanese population and first screening of 52 tag single nucleotide polymorphisms (SNPs). In the upper panel, P values for differences in allele frequencies were calculated by chi-square test (more ...)
Among the tag SNPs, rs10168266C>T, rs11889341C>T, and rs7574865G>T were most significantly associated with SLE in the first screening (P < 0.01). Allele frequencies of rs10168266T, rs11889341T, and rs7574865T were significantly increased in SLE compared with healthy controls (Table and Figure ). These SNPs were located in the introns of STAT4 and in LD with each other. In contrast, significant association was not detected for SNPs in the STAT1 region (P > 0.05).
Minor allele frequencies and P values for 52 tag single nucleotide polymorphisms in the STAT1-STAT4 region in the first screening
To confirm the association detected in the first screening, additional patients and controls were genotyped for the three SNPs using the TaqMan SNP Genotyping Assay, and association was examined in 308 SLE patients and 306 healthy controls in total (Table ). Significant deviation from Hardy-Weinberg equilibrium was not detected in healthy controls (P > 0.05). The rs7574865T allele, previously shown to be associated with SLE in Caucasians, was significantly increased in SLE patients (46.3%) compared with controls (33.5%, P = 4.9 × 10-6, OR 1.71). The association was compatible with the dominant model, under which the OR was 2.19 (T/T + G/T versus G/G).
Association of STAT4 single nucleotide polymorphisms rs10168266, rs11889341, and rs7574865 with systemic lupus erythematosus
The SNPs rs11889341 and rs10168266 were in LD with rs7574865 (r2: 0.57 to 0.78, D': 0.91 to 0.97) and were also significantly associated with SLE (allele frequency: P = 6.6 × 10-6 and P = 6.3 × 10-6, respectively). Haplotype analysis revealed that the haplotype carrying rs10168266T, rs11889341T, and rs7574865T was significantly increased (SLE: 36.8%, control: 24.3%, P = 1.5 × 10-6) whereas the haplotype carrying 10168266C, rs11889341C, and rs7574865G was significantly decreased in SLE (SLE: 52.7%, control: 65.0%, P = 1.0 × 10-5). Logistic regression analysis demonstrated that the association of each SNP lost statistical significance when adjusted for genotype of the other SNPs (Table ). Thus, due to the strong LD, it was impossible to identify a single causative SNP among the three.
Logistic regression analysis of the systemic lupus erythematosus-associated single nucleotide polymorphisms in STAT4
We next tested whether STAT4
rs7574865 was associated with phenotypes of SLE such as presence of nephritis, anti-dsDNA antibodies, and early age of onset (less than 20 years) as STAT4
genotype has been shown to be more strongly associated with subgroups of SLE with these phenotypes [10
] (Table ). Association of rs7574865 was observed both in SLE patients with nephritis (P
= 1.0 × 10-5
, OR = 1.85) and in those without nephritis (P
= 0.0031, OR = 1.55). The association was stronger in SLE patients with nephritis, although the difference between SLE with and without nephritis (case-only analysis) did not reach statistical significance. Similarly, rs7574865T was significantly increased in SLE patients with anti-dsDNA antibodies compared with healthy controls, whereas association was not detected in SLE patients without anti-dsDNA antibodies. The frequency of rs7574865T was slightly higher in the patients with an age of onset of less than 20 years as compared with greater than or equal to 20 years, although the difference was not statistically significant. These tendencies are consistent with those reported in Caucasians [10
]. These interpretations were not affected when the significance level was corrected for the number of comparisons (three phenotypes).
Association of STAT4 rs7574865 with characteristics of systemic lupus erythematosus such as nephritis, age of onset, and anti-double-stranded-DNA antibodies
To evaluate the epidemiological significance of STAT4
polymorphism in the genetic background of SLE in the Japanese population, we estimated the PAR% in Japanese persons and Caucasians using our present data and previously reported data [8
] (Table ). Because the frequency and OR of the risk genotype of rs7574865 were greater in the Japanese population than those of North Americans of European descent [8
], PAR% in the Japanese population (40.2%) was much higher than that of the latter (19.5%). A similarly high PAR% was observed in two of the three Japanese case-control series reported by Kobayashi and colleagues [12
] and in Colombians [11
]. Because PAR% may be affected by the difference in the method of ascertainment of each study, this comparison may not be completely valid. Nevertheless, these observations suggested that the contribution of STAT4
for SLE is greater in the Japanese population as compared with the Americans of European descent.
Population attributable risk percentage of STAT4 rs7574865 under the dominant model
At this point, molecular mechanisms that account for the association of STAT4
intron SNPs with SLE remain unclear. Studies with lupus model mice lacking Stat4
showed conflicting results. Stat4
deficiency reduced nephritis and autoantibody production in B6.NZM.Sle1.Sle2.Sle3
]. In contrast, Stat4
-deficient NZM (New Zealand mixed) mice developed accelerated nephritis and increased mortality in the absence of high levels of autoantibodies [25
]. STAT4 has been shown to be involved in the induction of IFNγ, differentiation of Th1 and Th17 cells, and signal transduction from type I IFN receptors [15
]. Th1 cytokines, especially IFNγ, have been shown to play a role in the pathogenesis of lupus nephritis [26
]. Recently, T cells from SLE patients were shown to produce excessive amounts of IFNγ upon stimulation [27
]. These observations may implicate the role of STAT4
SNPs in IFNγ production.
The role of type I IFNs in SLE has been established [1
]. Elevated serum type I IFN levels and expression of IFN-inducible genes in peripheral mononuclear cells were reported in SLE [28
]. The association of IRF5
, which induces type I IFNs, with SLE has been established [2
]. STAT4 is activated by type I IFN as well as IL-12 signals and produces IFNγ [15
]. Thus, STAT4 may also contribute to SLE as a component of the type I IFN signal pathway. Furthermore, STAT4 has been reported to transduce IL-12 signals to induce IFNγ production in B cells [30
It is interesting to note that significant association of STAT4 was not observed in SLE patients without anti-dsDNA antibodies (Table ). It would have been interesting to examine the effect of the genotype on the levels, rather than presence or absence, of anti-dsDNA antibody However, because the antibody levels fluctuate in association with disease activity and treatment, association with the genotype should be examined using the lifetime highest anti-dsDNA antibody level of each patient. Such data were not available for this study, and we hope that we can address this issue in the future.
Most of these observations imply that STAT4
risk genotype may be associated with an elevated expression level and/or function of STAT4 protein. A recent study reported that the STAT4
risk allele was associated with overexpression of STAT4
in osteoblasts but not in B cells [13
]. To address the significance of such findings, it will be necessary to examine the effect of this genotype on the expression levels and splicing isoforms in T and B cells.