The Minor Allele of rs7574865 in the STAT4 Gene Is Associated with Increased mRNA and Protein Expression

Abstract

Objective: The T allele of rs7574865 in STAT4 confers risk of developing autoimmune disorders. However, its functional significance remains unclear. Here we analyze how rs7574865 affects the transcription of STAT4 and its protein expression.

Methods: We studied 201 patients (80% female; median age, 54 years; median disease duration, 5.4 months) from PEARL study. Demographic, clinical, laboratory and therapeutic data were collected at each visit. IL-6 serum levels were measured by enzyme immune assay. The rs7574865 was genotyped using TaqMan probes. The expression levels of STAT4 mRNA were determined at 182 visits from 69 patients using quantitative real-time polymerase chain reaction. STAT4 protein was assessed by western blot in 62 samples from 34 patients. To determine the effect of different variables on the expression of STAT4 mRNA and protein, we performed multivariate longitudinal analyses using generalized linear models.

Results: After adjustment for age, disease activity and glucocorticoid dose as confounders, the presence of at least one copy of the T allele of rs7574865 was significantly associated with higher levels of STAT4 mRNA. Similarly, TT patients showed significantly higher levels of STAT4 protein than GG patients. IL-6 induced STAT4 and STAT5 phosphorylation in peripheral blood lymphocytes. Patients carrying at least one T allele of rs7574865 displayed lower levels of serum IL-6 compared to GG homozygous; by contrast the production of C-reactive protein was similar in both populations.

Conclusion: Our data suggest that the presence of the rs7574865 T allele enhances STAT4 mRNA transcription and protein expression. It may enhance the signaling of molecules depending on the STAT4 pathway.

Fig 1. Effect of rs7574865 polymorphism in the STAT4 mRNA levels of patients with early arthritis.

(A) Expression levels of STAT4 mRNA during follow-up. Significance level was determined through the Cuzic’s nonparametric test for trend across ordered groups. Statistical significance was considered p<0.05. (B) Data are clustered according to the genotype of rs7574865 in STAT4, homozygote GG (white boxes) versus presence of the T allele (gray boxes), and visits during follow-up. Data are presented as the interquartile range (p75 upper edge, p25 lower edge, p50 midline), p95 (line above the box), and p5 (line below the box) of relative STAT4 mRNA expression (2^-ΔΔCt). Dots represent the outliers.

Fig 2. STAT4 mRNA is increased in patients with active early arthritis carrying the T allele of rs7574865.

Expression levels of STAT4 mRNA according to the genotype of rs7574865, homozygote GG (white boxes) versus presence of the T allele (gray boxes), and disease activity level based on DAS28. Data are presented as the interquartile range (p75 upper edge, p25 lower edge, p50 midline), p95 (line above the box), and p5 (line below the box) of relative mRNA STAT4 expression (2^-ΔΔCt). Dots represent the outliers. Significance level was determined through the Mann-Withney test. Due to multiple comparisons, statistical significance was considered p<0.0125 (Bonferroni correction).

Fig 3. STAT4 protein is increased in patients with early arthritis carrying the TT genotype of rs7574865.

A) Western blot analysis of the STAT4 expression in PBMCs from patients with TT or GG rs7574865 genotype with different treatments. Representative blots are shown. B and C) Densitometric quantification of STAT4 protein expression normalized to β-actin expression. The graph represents the linear prediction with 95% confidence intervals of STAT4/β-actin ratio according to the multivariable analysis displayed in Table 3 represented by rs7574865 genotype (B) or by DMARD treatment (No DMARD, Monotherapy or Combined Therapy) and rs7574865 genotype (C).

Fig 4. Interleukin-6 induces STAT4 phosphorylation.

(A) Representative flow cytometry dot plots showing the expression of phosphoSTAT4 or phosphoSTAT5 positive cells on PBLs unstimulated or stimulated with 10 ng/ml IL-6, 50 ng/ml IL-12 or 100 U/ml IL-2 for 15 min. The percentage of each population is indicated. (B) Percentage of phosphoSTAT4 (upper panel) or phosphoSTAT5 (lower panel) positive PBLs unstimulated (basal) or stimulated with 10 ng/ml IL-6, 50 ng/ml IL-12 or 100 U/ml IL-2 for 15 min. Data are represented as the mean ± sem of 20 patients from PEARL study. Black bars represents patients with GG genotype and grey bar patients with TT genotype for rs7574865; no significant differences were obtained between them (Mann-Whitney test).

Fig 5. Interleukin-6 (IL-6) induces higher levels of C reactive protein in patients with, at least one T allele of rs7574865.

IL-6 (panel A) and C-reactive protein (B panel) serum levels in patients with early arthritis clustered by disease activity level and rs7574865 genotype (white boxes GG genotype, grey boxes GT and TT genotypes). Data are shown as the interquartile range (p75 upper edge of the box, p25 lower edge, p50 midline), as well as the p95 (line above the box) and p5(line below the box). Dots represent outliers. C) Correlation between IL-6 levels and C-reactive protein production depending of rs7574865 genotype. Data are shown as the fitted linear prediction and its 95% confidence interval (clear grey shadow GG genotype, dark grey shadow GT and TT genotypes) using the twoway command with the lfitci option.