The HBV natural clearance subjects and ASCs were revisited during the follow-up in 2011. Those whose diagnoses were the same as the previous examinations were finally involved in this study. Patients with chronic hepatitis B (CHB), HBV-infected patients with liver cirrhosis (LC), and HBV-infected patients with HCC were Carfilzomib recruited from Changzheng Hospital, Changhai Hospital, and Eastern Hepatobiliary Surgery Hospital of this university, Southwest hospital, Chongqing, and the 88th hospital in Taian city, Shandong, China, from October 2009 to September 2011. ASC status, CHB, LC, and HCC were diagnosed according to the criteria as previously described [35], [36]. The subjects seropositive for antibodies to HCV or hepatitis delta virus were not included.

A total of 1,012 healthy controls, 302 HBV natural clearance subjects, 316 ASCs, 316 patients with CHB, 358 HBV-infected patients with LC, and 1,021 HBV-infected patients with HCC were enrolled in this study. The study subjects were of Han Chinese ancestry. Ethics Statement A written consent was obtained from each study subject. The study protocol conformed to the 1975 Declaration of Helsinki and was approved by the ethics committee of Second Military Medical University. Serological Viral Parameters Examination, HBV Genotyping, and Mutation Analysis HBsAg, anti-HBs, hepatitis B e antigen (HBeAg), anti-HBe, anti-HBc, HBV DNA, anti-HCV, alpha-fetoprotein, liver function parameters including alanine aminotransferase (ALT) were examined as previously described [13], [35], [36].

Antibody to hepatitis delta virus was examined using commercial kits (Wantai Bio-Pharm, Beijing, China). HBV genotyping, PCR amplification of the HBV EnhII/BCP/PC region and the preS region, and viral mutation analysis were carried out as previously reported [35], [37], [38]. Genotyping of the SNPs The SNPs were genotyped using fluorescent-probe real-time quantitative PCR in a LightCyclerTM480 (Roche, Basel, Switzerland). Probes (Minor Groove Binder [MGB]) and primers were commercially designed and synthesized (GeneCore BioTechnologies, Shanghai, China). Sequences of the primers and probes and PCR amplification condition are listed in Table S1. Each reaction mixture contained 0.2 ��mol/L of primers and probes, 1�C4 ng/��L purified templates in Premix Ex Taq reaction system (Takara, Dalian, China).

The genotyping was performed without knowing the participants�� disease status. Two blank controls in each 96-well format were used for quality control, and more than 10% of samples were randomly selected to repeat, yielding a 100% concordance. The success rates of genotyping for all of these SNPs were greater than 99%. Statistical Analysis Hardy-Weinberg equilibrium (HWE) of each Dacomitinib SNP in the study population was examined online (http://ihg.