Association of STAT4 Polymorphisms with Susceptibility to Type-1 Autoimmune Hepatitis in the Japanese Population

Background/Aims Recent studies demonstrated an association of STAT4 polymorphisms with autoimmune diseases including systemic lupus erythematosus and rheumatoid arthritis, indicating multiple autoimmune diseases share common susceptibility genes. We therefore investigated the influence of STAT4 polymorphisms on the susceptibility and phenotype of type-1 autoimmune hepatitis in a Japanese National Hospital Organization (NHO) AIH multicenter cohort study. Methodology/Principal Findings Genomic DNA from 460 individuals of Japanese origin including 230 patients with type-1 autoimmune hepatitis and 230 healthy controls was analyzed for two single nucleotide polymorphisms in the STAT4 gene (rs7574865, rs7582694). The STAT4 rs7574865T allele conferred risk for type-1 autoimmune hepatitis (OR = 1.61, 95% CI = 1.23–2.11; P = 0.001), and patients without accompanying autoimmune diseases exhibited an association with the rs7574865T allele (OR = 1.50, 95%CI = 1.13–1.99; P = 0.005). Detailed genotype-phenotype analysis of type-1 autoimmune hepatitis patients with (n = 44) or without liver cirrhosis (n = 186) demonstrated that rs7574865 was not associated with the development of liver cirrhosis and phenotype (biochemical data and the presence of auto-antibodies). Conclusions/Significance This is the first study to show a positive association between a STAT4 polymorphism and type-1 autoimmune hepatitis, suggesting that autoimmune hepatitis shares a gene commonly associated with risk for other autoimmune diseases.


Introduction
Autoimmune hepatitis (AIH) is characterized by chronic inflammation of the liver, interface hepatitis, hypergammaglobulinemia and production of autoantibodies [1,2]. The etiology of AIH is unknown, but is thought to have both a genetic and an environmental basis [3]. Although the HLA DRB1 gene is a wellcharacterized susceptibility gene [4,5], non-HLA susceptibility genes may also contribute to genetic susceptibility to AIH and remain to be elucidated. Recently, with the emergence of genomewide association studies (GWAS), there has been a dramatic increase in genetic discoveries for many complex genetic autoimmune diseases, such as type 1 diabetes and rheumatoid arthritis (RA) [6]. It is also interesting to note that evaluating the results from the study of one disease in other complex diseases can disclose common risk factors. Thus, there has been a marked overlap of loci between autoimmune diseases [7]. Of those, STAT4 particularly has been confirmed in several studies and is clearly associated with autoimmune diseases such as RA or systemic lupus erythematosus (SLE) [8][9][10]. STAT4, a signal transducer and activator of transcription 4, is expressed in activated peripheral blood monocytes, dendritic cells and macrophages at the sites of inflammation in humans [11]. It is activated by interleukin (IL)-12, leading to T helper (Th) 1 and Th 17 differentiation, monocyte activation and interferon (IFN)-a production [12]. Since Th1 and Th17 cells have the capacity to cause autoimmunity [13], STAT4 may play a crucial role in the development of autoimmune diseases, including AIH.
The degree of risk for RA or SLE susceptibility observed with the STAT4 haplotype was found to be similar in Caucasian and Japanese populations [14][15][16]. In addition, meta-analysis demonstrated that the STAT4 rs7574865 T allele conferred susceptibility to various autoimmune diseases, suggesting an association between STAT4 gene polymorphism and autoimmune diseases [17].
STAT4 is considered important in a mouse model of Th1dependent liver injury [18]. Therefore, we hypothesized that STAT4 polymorphisms may overlap in genetic susceptibility between AIH and other autoimmune diseases. To test this hypothesis, we investigated the association of STAT4 with type-1 AIH susceptibility using a large series of Japan NHO-AIH registry [19]. We also tried to evaluate whether the gene was associated with type-1 AIH outcome measures in a Japanese AIH cohort.

Study population
Consecutive type-1 AIH patients were initially enrolled in the register of the Japanese National Hospital Organization (NHO) liver-network study, contributed to medical facilities in Japan, and prospectively followed since 2009 as a multicenter cohort population. All patients satisfied the 1999 revised criteria of International Autoimmune Hepatitis Group (IAIHG) diagnosis of type-1 AIH [20]. Patients were excluded from the study if there was histological evidence of cholangitis or non-alcoholic steatohepatitis. In addition, patients who were positive for hepatitis B virus (HBV)-surface antigen (HBsAg) or hepatitis C virus (HCV)-RNA were excluded. Patients with other causes of liver disease, such as excess alcohol or drug use, were excluded based on reviews of their appropriate history and investigations. The control group consisted of 230 gender-matched Japanese healthy subjects (34 men and 196 women

Variables at study entry
Demographic and other characteristics of the 230 retained patients were recorded in a database at the initial assessment. Data included sex, age at diagnosis, time of onset of symptoms or other evidence of liver disease, markers of infection with hepatitis viruses HBV and HCV, alcohol intake, coexisting autoimmune diseases, serum levels of ALT, AST, alkaline phosphatase and bilirubin, platelet count and prothrombin time. Anti-nuclear antibodies (ANA) and anti-smooth muscle antibodies (ASMA) were measured by indirect immunofluorescence on HEp-2 cells and cut-off titers for positivity were 1:40. Liver tissue from percutaneous biopsy performed at the referring facility was available for the majority of patients at the time of entry (192/230, 83.5%), but for only a few at the subsequent follow-up examination (7/230, 3.0%). The histological variables examined included degree of fibrosis (0; absent, 1; expansion of fibrosis to parenchyma, 2; portal-central or portal-portal bridging fibrosis, 3; presence of numerous fibrous septa, 4; multi-nodular cirrhosis). The histological diagnosis of cirrhosis required a loss of the normal lobular architecture, reconstruction of hepatic nodules and presence of regenerative nodules [21]. Liver biopsy was not performed for patients who had apparent biochemical, endoscopic and ultrasound features of liver cirrhosis. All phenotypic data were collected blind to the results of the genotypic data.
Rs7574865 was a 147-bp PCR product and was digested with restriction enzyme HpaI (New England Biolabs) and electrophoresed on a 2.5% polyacrylamide gel. Rs7574865 was a 338-bp PCR product was digested with restriction enzyme HpyCH4III (New England Biolabs) and electrophoresed on a 3.0% polyacrylamide gel.
HLA-DRB1 genotyping was performed as described previously [24]. Briefly, the HLA-DRB1 genotype was determined by sequence-based typing (SBT) of group-specific PCR products.

Statistical analyses
Results are expressed as mean 6 SD. The statistical significance of differences between groups was calculated by either the chisquare test or Fisher's exact test for categorical data and Mann-Whitney's U-test for quantitative data. Multivariate logistic regression analysis was performed with SPSS v.18 for windows (SPSS Statistics, Illinois). Deviation from Hardy-Weinberg equilibrium was assessed using the SNPAlyze software ver. 7.0 (Dynacom, Yokohama, Japan). Power calculations were performed by using an online power calculator [25]. A P value of ,0.05 was considered significant.

Baseline data at entry
Of the original 240 patients registered in the NHO-AIH study, 10 were excluded from analysis because of overlapping primary biliary cirrhosis (PBC). The remaining 230 patients were eligible for the study. Table 1 shows other demographic data for the cohort at entry. Among the enrolled type-1 AIH patients, 206 (89.6%) were positive for ANA (.1:40) and 96 (41.7%) for ASMA (.1:40). Some patients with lower serum aminotransferase or total bilirubin were managed with ursodeoxycholic acid (UDCA) therapy alone, which was demonstrated to be efficacious in Japanese patients with type I autoimmune hepatitis [26]. Among Association of STAT4 polymorphisms with type-1 AIH The genotype frequencies for STAT4 rs7574865 and rs7582694 were in HWE (Hardy-Weinberg equilibrium) in both the patient and control populations (data not shown). Because of the strong linkage disequilibrium between rs7574865 and rs7582694 (R 2 = 0.949 and D' = 0.981), very similar results were observed between rs7574865 (Table 2) and rs7582694 (Table 3). We observed a significant difference in allele frequency and genotype distribution of STAT4 polymorphisms (rs7574865) between type-1 AIH patients and controls. As shown in Table 2, the minor T allele and TT genotype frequencies at STAT4 rs7574865 in the type-1 AIH group differed significantly from those in the control group.
To determine whether the observed association of the STAT4 gene SNPs with disease susceptibility was caused by other autoimmune diseases associated with AIH, we stratified type-1 AIH patients without other overlapping autoimmune diseases. There was a significant association of STAT4 rs7574865 with susceptibility to type-1 AIH even in the AIH patients without other overlapping autoimmune diseases (Table 4).

Associations between STAT4 genotype status and type-1AIH phenotype
To examine the associations between HLA-DR and type-1 AIH, HLA-DR allele typing was performed in patients with type-1 AIH. In the analysis of HLA-DR alleles, the frequencies of DR *04 allele was significantly increased in type-1 AIH patients as compared with those in controls ( Table 5). The STAT4 rs7574865 T allele and HLA-DR *04 allele for the progression to liver cirrhosis were subjected to multivariate logistic regression analysis. Neither HLA-DR *04 allele nor rs7574865 T allele did not contribute to the progression to liver cirrhosis (data not shown). Based on the significant association of the rs7574865 with susceptibility to type-1 AIH, we also performed a detailed genotype-phenotype analysis using the clinical data. However, we found no significant difference in the presence of autoantibodies (ANA or ASMA) and the peak levels of transaminases or total bilirubin (AST, ALT, TB) by laboratory tests among each genotype (data not shown).   Discussion AIH reflects a complex interaction between triggering factors, environmental factors, genetic predisposition and the immune regulatory network [3]. Most knowledge concerning the genetic factors of AIH comes from studies of the HLA genes [4,5]. Although multiple genes are probably involved, HLA genes appear to play a dominant role in the predisposition to AIH [27]. Genetic factors other than HLA genes that can affect the susceptibility of AIH are mainly polymorphisms in genes that encode proteins that affect cytokine pathways responsible for modulating immunity [27][28][29]. Although autoimmune diseases include a wide array of different organ involvement and symptoms, they all share a common component: the loss of immune tolerance toward ''self antigen'' [30]. Findings in recent genetic studies support the emerging concept that distinct clinical autoimmune diseases may share genetic susceptibility factors. STAT4 is a critical transcription factor involved in the regulation of Th1/Th2 cytokine balance [12]. STAT4 polymorphisms have been found to be associated with various autoimmune diseases [8][9][10].
This study is the first to investigate a detailed correlation between STAT4 gene polymorphisms and susceptibility to type-1 AIH in a Japanese nationwide AIH cohort study. In the current study, we confirmed an association of STAT4 polymorphisms with susceptibility to type-1 AIH. Our data suggest that STAT4 may be an ''autoimmune disease susceptibility gene'' and support the concept of deregulated pathways across multiple autoimmune diseases. In addition to their influence on autoimmune disease susceptibility, STAT4 polymorphisms can also influence disease phenotypes. For example, rs7574865 in SLE patients was associated with severe disease manifestations, such as nephritis, high double stranded-DNA antibody production and younger age of disease onset. [31] For patients with systemic sclerosis, this polymorphism was associated with the presence of pulmonary  fibrosis [32]. Therefore, we examined possible associations between STAT4 and the clinical phenotype of type-1 AIH. However, we did not find evidence of association between STAT4 polymorphisms and disease progression or phenotype of type-1 AIH.
Regarding the disease-developing effect of genetic variants in the STAT4 region on type-1 AIH observed in our study, it might be interesting to determine whether the STAT4 risk alleles have different expression levels or functional effects in different effector cells [33]. The susceptibility SNP rs7574865 is located within intron 3 of STAT4, a non-coding region. It is suspected that it may influence the gene expression of STAT4 at the level of transcription or splicing variation [34]. A recent study reported that the expression level of STAT4 in peripheral blood mononuclear cells correlated with the risk allele of STAT4 rs7574865 [33]. This might indicate the effects of different STAT4 gene variants on STAT4 expression levels. To date, the main alternative spliced isoforms of STAT4 are STAT4a and STAT4b. STAT4b is a shorter form of the full-length STAT4a and is not as efficient as STAT4a for the direct induction of IFN-c gene expression activated by IL-12 in Th1 cells [35]. However, expression of STAT4b, lacking the transactivation domain, was not affected by the STAT4 SNPs [33]. Additionally, a significant inverse correlation with T-risk alleles at rs7574865 and the methylation status of the STAT4 promoter was demonstrated in inflammatory bowel disease [36]. The STAT1 gene is located adjacent to STAT4 suggesting it is also a candidate susceptibility gene for autoimmune disease [37]. To examine the role of the STAT1-STAT4 region, 52 tag SNPs encompassing this region in Japanese lupus patients [38]. The SNPs rs11889341 and rs10168266 were in linkage disequilibrium (LD) with rs7574865 and were significantly associated with SLE [38]. In contrast, significant association was not detected for SNPs in the STAT1 region [38].
AIH pathogenesis are more complex than the traditional dichotomous Th1/Th2 paradigm, where STAT4 represents a transcription factor that induces IL-12, IL-23 and type 1 IFNmediated signals to Th1 and Th17 differentiation, monocyte activation and interferon-c production [39]. STAT4 is important for IL-22 production, which plays a pathological role in IL-17dependent hepatitis [40].
A recent study showed that G allele at rs7574865 was associated with increased risk for HCC, suggesting dual roles of STAT4 in autoimmune diseases and HBV-related HCC [41]. Interestingly, subjects with GG genotype at rs7574865 had the lowest mRNA levels of STAT4 in both HCC and non-tumor tissues compared with TG and TT genotypes [41]. Considering the role of STAT4 in Th1 immune responses, rs7574865 polymorphisms may affect the hepatic immune response against auto-antigen or viral antigen, contributing to the susceptibility of these related disorders. Further studies will be needed to examine the different possible mechanisms by which the variant haplotypes contribute to AIH.
The current study was limited because there were relatively small numbers of patients, and because some of the phenotypes examined were related to disease activity, and therefore may have fluctuated naturally or as a result of treatment. Additionally, it was difficult to perform a replication study due to the very low prevalence of type-1 autoimmune hepatitis and limited numbers of enrolled patients. In the current study, the power to detect a 1.6fold increased risk, assuming an alpha value of 0.05, was 0.627 for rs7574865 T allele. Another limitation is the lack of complete information regarding the causal polymorphisms and their exact functional roles.
In summary, our results identified STAT4 SNP rs7574865 as a disease-susceptible gene variant in type-1 AIH. Further studies on the expression and regulation of STAT4 in the liver will be required to investigate the functional consequences of STAT4 gene variants in more detail.