| Background: Liver diseases are common in China. The major types of liver diseases are hepatitis B virus (HBV) related disorders (such as chronic hepatitis B, liver cirrhosis and hepatocellular carcinoma), it is different from liver disease spectrum in Caucasians, in which lipid/alcoholic hepatitis, drug induced hepatitis, hepatitis C and autoimmune liver diseases are more common. Beside this, hepatitis A/C/E virus infection also need to be attention. Followed with the improvement of diagnostics and emphasis level, the autoimmune liver diseases are highly recognized and treated in recent years. The prevalence and some clinical characteristics of autoimmune diseases are significantly different among different ethnic populations, e.g, combination autoimmune manifestion with other organs are rare in Chinese patients. However, the clinical characteristics and host genetic factors of autoimmune hepatitis have not been fully analysed.It is an important and challenging issue for identification of host genetic factors involved in viral hepatitis and autoimmune hepatitis, which has not been fully understand. For such common complex diseases, genetic association studies are widely used, based on case control design. Genetic associations are detected between biological functional candidate genes and diseases. T helper (Th) cells polarization play a key role in the pathogenetic process of viral infection and autoimmune hepatitis.T-bet is a member of the T-box family of transcription factors, which is responsible for the induction of Th1 cells and the repression of Th2 cells from naive T lymphocytes. Reduced T-bet concentrations have been linked with diminished responses to some viral infections, including human immunodeficiency virus (HIV) infection and woodchuck hepatitis virus (WHV) infection, and higher levels have been linked with inflammatory outcomes such as Th1-mediated colitis and liver injury in animal models. The human TBX21 gene encoding T-bet, some common single nucleotide polymorphisms (SNPs) are located in the promoter region. T-1993C is a functional site which may influence the transcriptional activity of TBX21 promoter.T-cell immunoglobulin domain and mucin domain protein 1 (TIM-1) is a glycoprotein, which is expressed on the surface of CD4+ Th cells. It plays an important role on the Th1/Th2 polarization. TIM-1 is highly expressed on the surface of Th2 cells as a co-stimulation molecule and induces the activation and proliferation of Th2 cells. TIM-1 is also the only cellular receptor for hepatitis A virus (HAV). TIM-1 is polymorphic in population, there is a 18-bp insertion variation in exon 4 (mucin domain).Objective: Because the expression of TBX21 and TIM-1 are important for Th1/Th2 immune response, it is interesting to examine whether polymorphisms of TBX21 and TIM-1 genes are associated with HAV/HBV infection and autoimmune hepatitis. In this study, we collected cases and controls samples based on hospital or community, then conducted genetic association studies between TBX21/TIM-1 genes and HAV infection, HBV infection and autoimmune hepatitis.Methods: We completed the following works:1. We newly collected 1758 cases and controls (1241 males and 517 females), including normal healthy controls, patients with chronic HBV infection or spontaneously clearance, patients with hepatitis A infection, cases with asymptomatic HAV infection, cases negative for HAV markers and patients with autoimmune hepatitis. Clinical and epidemiological information were collected, genomic DNA was extracted. Along with previous samples, we made DNA panels.2. Eight known SNPs of TBX21, the T-1993C, T-1514C, G-1499A, C99G, T1298C, G6547A, T7725C and T9903C, were validated in our representative samples, appropriate genotyping methods were developed.3. The polymorphisms in exon 4 of TIM-1 were discovered by TA-clone and sequencing, haplotypes were constructed, and the GeneScan genotyping method for 157MVTTTP insertion/deletion was established.4. TBX21 T-1993C and TIM-1 157MVTTTP insertion/deletion polymorphisms were selected for genotyping in normal healthy controls, patients with chronic HBV infection or spontaneously clearance, patients with hepatitis A infection, cases with asymptomatic HAV infection, cases negative for HAV markers and patients with autoimmune hepatitis. Genetic association analysis were conducted.Results: Based on studies mentioned above, we demonstrated the following main results:1. T-1993C is the most common polymorphism in the exons and promoter region of TBX21. Previously reported G-1499A and T9903C SNP had very low frequencies (< 1%) in Chongqing population.2. The exon 4 of TIM-1 is highly polymorphic in Chongqing population. There were four major haplotypes in Chongqing population.3. Compared with asymptomatic HBV carriers, TBX21 T-1993C was not associated with disease progression of chronic HBV infection. Compared with cases with asymptomatic HAV infection, TBX21 T-1993C was not associated with HAV infection.4. Compared with patients of chronic HBV infection, TBX21 T-1993C was significantly associated with spontaneously clearance of HBV (χ2 = 20.4, P = 0.0000063). Similarly, haplotype -1993T-1514T was associated with decreased susceptibility to the persistence of HBV infection (P = 0.000017, OR = 0.46, 95% CI 0.32-0.65).5. Compared with blood donors, TBX21 -1993T allele was significantly associated with autoimmune hepatitis (χ2 = 13.7, P = 0.00004). By logistic regression controlling confounding factors such as age and sex, -1993T/T homozygotes had a significantly increased risk for autoimmune hepatitis (P=0.00043, OR=0.26, 95%CI = 0.12-0.55).6. TIM-1 157MVTTTP insertion/deletion polymorphism had no significant influence on the genetic risks for HAV infection, HBV infection or autoimmune hepatitis.Conclusion: In conclusion, based on case control collection, polymorphic loci screening and genotyping method establishment, we selected TBX21 T-1993C and TIM-1 157MVTTTP insertion/deletion polymorphisms for genotyping in our large case control samples. We detected the genetic associations between TBX21/TIM-1 genes and HAV infection, HBV infection and autoimmune hepatitis. Our case control association study indicated that TBX21 T-1993C was associated with HBV clearance and autoimmune hepatitis. Previous luciferase reporter assays and electrophoretic mobility shift assays suggest T-1993C polymorphism affects the transcriptional activity of TBX21 and the -1993C allele may contribute to an increase in T-bet expression. Nevertheless, more steps are required before the importance of the TBX21 gene in HBV infection and autoimmune hepatitis can be fully ascertained. First, data from other ethnic populations are needed to confirm our initial observation. Second, the promoter polymorphisms in the TBX21 gene and its functional molecular mechanisms for this association with diseases should be identified.
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